CN118401658A - Methods of regulating PCSK9 and uses thereof - Google Patents

Abstract

The present disclosure provides CRISPR/Cas9-based fusion molecules and guide RNAs for targeted reduction or elimination of PCSK9 gene products in vivo. The present disclosure also relates to preparations, methods of production thereof, and methods of use thereof.

Description

Methods of regulating PCSK9 and uses thereof

Related Applications

This application claims priority and the benefits of PCT application No. PCT/CN2021/133681 filed on November 26, 2021, the contents of which are incorporated herein by reference in their entirety.

Incorporation by Reference to the Sequence Listing

The sequence listing XML associated with this application is provided electronically in XML file format and is incorporated into this specification by reference. The name of the XML file containing the sequence listing XML is "EPIG-001_001WO_SequenceListing_ST26". The size of this XML file is 220,215 bytes and was created on November 1, 2022.

Background technique

Engineered DNA-binding proteins that can be customized to target any gene in mammalian cells have enabled rapid advances in biomedical research and are an ideal platform for gene therapy. The RNA-guided CRISPR-Cas9 system has emerged as a promising platform for programmable targeted gene regulation. Fusion of a catalytically inactive "dead" Cas9 (dCas9) to a Kruppel-associated box (KRAB) domain generates synthetic repressors that are able to regulate or silence target genes with high specificity and potency in cell culture experiments.

However, the continuous regulation and silencing of endogenous genes using synthetic dCas9-KRAB fusion proteins poses challenges for in vivo therapeutic applications. Synthetic repressors exceed the packaging size limitations of viral vector delivery methods. Safety, toxicity, immunogenicity, and off-target effects are other challenges that limit the use of synthetic repressors in vivo. There is a need in the art for alternative methods for producing genetically engineered synthetic gene repressors and in vivo delivery of the synthetic gene repressors for use as therapeutic agents. The present disclosure addresses this unmet need in the art.

Summary of the invention

The present disclosure provides a method for regulating (e.g., reducing or eliminating) the expression of a proprotein convertase subtilisin/Kexin type 9 (PCSK9) gene product in a cell, the method comprising the step of introducing the following substances into the cell: a fusion molecule comprising at least one DNA binding protein and at least one gene expression regulator, or a nucleic acid sequence encoding the fusion molecule, wherein the gene expression regulator provides modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element, thereby regulating (e.g., reducing or eliminating) the expression of the PCSK9 gene product in the cell.

The present disclosure provides an in vivo method for regulating (e.g., reducing or eliminating) the expression of a PCSK9 gene product in a subject, the method comprising the step of introducing the following substances into the cells of the subject: a fusion molecule comprising at least one DNA binding protein and at least one gene expression regulator, or a nucleic acid sequence encoding the fusion molecule, wherein the gene expression regulator provides modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element, thereby regulating (e.g., reducing or eliminating) the expression of the PCSK9 gene product in the subject.

The present disclosure provides a method for regulating (e.g., reducing) low-density lipoprotein (LDL) cholesterol in a subject, the method comprising the step of introducing the following substances into the cells of the subject: a fusion molecule comprising at least one DNA binding protein and at least one gene expression regulator, or a nucleic acid sequence encoding the fusion molecule, wherein the gene expression regulator provides modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element, thereby regulating (e.g., reducing) the LDL cholesterol in the subject.

The present disclosure provides a method for treating or alleviating the symptoms of a PCSK9-related disease in a subject, the method comprising the step of introducing the following substances into the cells of the subject: a fusion molecule comprising at least one DNA binding protein and at least one gene expression regulator, or a nucleic acid sequence encoding the fusion molecule, wherein the gene expression regulator provides modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element, thereby treating or alleviating the symptoms of the PCSK9-related disease in the subject.

The present disclosure provides a method for amplifying a cell population with reduced expression of a PCSK9 gene product, the method comprising the following steps: i) introducing a fusion molecule comprising at least one DNA binding protein and at least one gene expression regulator or a nucleic acid sequence encoding the fusion molecule into a plurality of cells, wherein the gene expression regulator provides a modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element; ii) amplifying the plurality of cells to produce a plurality of modified cells with a stable reduction in expression of a PCSK9 gene product, wherein the expression of the PCSK9 gene product of the plurality of modified cells is reduced by at least 50%, at least 60%, at least 70%, at least 80% or at least 90% relative to cells into which the gene expression regulator is not introduced, and wherein the cells are hepatocytes. In certain embodiments, the reduction in expression of the PCSK9 gene product is a transient reduction. In certain embodiments, the reduction in expression of the PCSK9 gene product is a stable reduction.

In certain embodiments, the PCSK9 regulatory element is a core promoter, a proximal promoter, a distal enhancer, a silencer, an insulator element, a boundary element, or a locus control region.

In some embodiments, the modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element is located within about 100bp, about 200bp, about 300bp, about 400bp, about 500bp, about 600bp, about 700bp, about 800bp, about 900bp, about 1000bp, about 1100bp, about 1200bp, about 1300bp, about 1400bp or about 1500bp upstream of the transcription start site of the PCSK9 gene. In some embodiments, the modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element is located within 1000bp upstream of the transcription start site of the PCSK9 gene. In some embodiments, the modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element is located within 300bp upstream of the transcription start site of the PCSK9 gene.

In some embodiments, the modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element is located within about 100bp, about 200bp, about 300bp, about 400bp, about 500bp, about 600bp, about 700bp, about 800bp, about 900bp, about 1000bp, about 1100bp, about 1200bp, about 1300bp, about 1400bp or about 1500bp downstream of the transcription start site of the PCSK9 gene. In some embodiments, the modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element is located within about 300bp downstream of the transcription start site of the PCSK9 gene. In some embodiments, the modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element is located within 1000bp upstream of the transcription start site of the PCSK9 gene and within 300bp downstream of the transcription start site.

In certain embodiments, the modification of the at least one nucleotide is DNA methylation.

In certain embodiments, the at least one gene expression regulator comprises a DNA methyltransferase (DNMT), a DNA demethylase, a histone methyltransferase, a histone demethylase, or a portion thereof.

In certain embodiments, the at least one gene expression regulator comprises a DNA methyltransferase (DNMT) or a portion thereof. In certain embodiments, the DNA methyltransferase is DNMT3A, DNMT3B, DNMT3L, DNMT1 or DNMT2. In certain embodiments, the DNMT3A comprises the amino acid sequence of SEQ ID NO: 23. In certain embodiments, the DNMT3L comprises the amino acid sequence of SEQ ID NO: 24.

In certain embodiments, the at least one gene expression regulator comprises a zinc finger protein-based transcription factor or a portion thereof. In certain embodiments, the zinc finger protein-based transcription factor is a Kruppel-associated repression cassette (KRAB). In certain embodiments, the KRAB comprises the amino acid sequence of SEQ ID NO: 22.

In some embodiments, the at least one gene expression regulator comprises a DNA methyltransferase or a portion thereof and a zinc finger protein-based transcription factor or a portion thereof. In some embodiments, the DNA methyltransferase is selected from DNMT3A and DNMT3L and a combination thereof, and the zinc finger protein-based transcription factor is KRAB.

In some embodiments, the at least one DNA binding protein is Cas9, dCas9, Cpf1, zinc finger nuclease (ZNF), transcription activator-like effector nuclease (TALEN), homing endonuclease, dCas9-FokI nuclease or MegaTal nuclease. In some embodiments, the at least one DNA binding protein is dCas9. In certain embodiments, the dCas9 comprises Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, Campylobacter jejuni dCas9, Corynebacterium diphtheria dCas9, Eubacterium ventriosum dCas9, Streptococcus pasteurianus dCas9, Lactobacillus farciminis dCas9, Sphaerochaeta globus dCas9, Azospirillum (e.g., strain B510) dCas9, Gluconacetobacter diazotrophicus dCas9, Neisseria cinerea dCas9, Roseburia enterica dCas9, and Streptococcus pasteurianus dCas9. intestinalis) dCas9, Parvibaculum lavamentivorans) dCas9, Nitratifractor salsuginis (e.g., strain DSM 16511) dCas9, Campylobacter lari (e.g., strain CF89-12) dCas9, Streptococcus thermophilus (e.g., strain LMD-9) dCas9. In some embodiments, the dCas9 comprises the amino acid sequence of SEQ ID NO: 1.

In certain embodiments, the fusion molecule comprises the at least one gene expression regulator fused to the C-terminus, the N-terminus, or both, of the at least one DNA binding protein.

In some embodiments, the at least one gene expression regulator is directly fused to the at least one DNA binding protein. In some embodiments, the at least one gene expression regulator is indirectly fused to the at least one DNA binding protein through a non-regulator, a second regulator, or a linker. In some embodiments, the fusion molecule comprises a dCas9 fused to KRAB on the C-terminal end and to DNMT3A and DNMT3L on the N-terminal end. In some embodiments, the fusion molecule comprises the amino acid sequence of SEQ ID NO: 97.

In some embodiments, the fusion molecule further comprises at least one nuclear localization sequence. In some embodiments, the at least one nuclear localization sequence is directly fused to the C-terminus, N-terminus, or both of the at least one DNA binding protein. In some embodiments, the at least one nuclear localization sequence is indirectly fused to the C-terminus, N-terminus, or both of the at least one DNA binding protein via a linker.

In some embodiments, the nucleic acid sequence encoding the fusion molecule is deoxyribonucleic acid (DNA). In some embodiments, the nucleic acid sequence encoding the fusion molecule is messenger ribonucleic acid (mRNA).

In some embodiments, the method further comprises the step of introducing at least one single guide RNA (sgRNA) or DNA encoding the sgRNA, wherein the sgRNA is complementary to a DNA sequence near the PCSK9 gene and/or within the PCSK9 regulatory element, thereby targeting the fusion molecule to the PCSK9 gene or PCSK9 regulatory element. In some embodiments, the sgRNA comprises a nucleic acid sequence of SEQ ID NO: 27-95 or 98-108.

In some embodiments, the fusion molecule is formulated in a liposome or lipid nanoparticle. In some embodiments, the fusion molecule and the sgRNA are formulated in a liposome or lipid nanoparticle. In some embodiments, the fusion molecule and the sgRNA are formulated in the same liposome or lipid nanoparticle. In some embodiments, the fusion molecule and the sgRNA are formulated in different liposomes or lipid nanoparticles.

In certain embodiments, the liposome or lipid nanoparticle comprises an ionizable lipid (20%-70%, molar ratio), a PEGylated lipid (0%-30%, molar ratio), a supporting lipid (5%-50%, molar ratio) and cholesterol (10%-50%, molar ratio). In certain embodiments, the ionizable lipid is selected from a pH-responsive ionizable lipid, a thermally responsive ionizable lipid and a light-responsive ionizable lipid.

In some embodiments, the fusion molecule is formulated in an AAV vector. In some embodiments, the fusion molecule and the sgRNA are formulated in an AAV vector. In some embodiments, the fusion molecule and the sgRNA are formulated in the same AAV vector. In some embodiments, the fusion molecule and the sgRNA are formulated in different AAV vectors.

In certain embodiments, the fusion molecule is delivered to the cell by local injection, systemic infusion, or a combination thereof.

In certain embodiments, the subject is a human.

In certain embodiments, the PCSK9-related disease is atherosclerotic cardiovascular disease. In certain embodiments, the PCSK9-related disease is hypercholesterolemia. In certain embodiments, the cell is a hepatocyte.

The present disclosure provides an sgRNA comprising a nucleic acid sequence of any one of SEQ ID NOs: 27-95 or 98-108. The present disclosure provides a DNA sequence encoding any one of the sgRNAs disclosed herein.

The present disclosure provides a pharmaceutical composition, comprising: a fusion molecule comprising at least one DNA binding protein and at least one gene expression regulator, or a nucleic acid sequence encoding the fusion molecule, wherein the fusion molecule targets a genomic region near a PCSK9 gene and/or within a PCSK9 regulatory element, wherein the at least one gene expression regulator provides modification of at least one nucleotide near the PCSK9 gene and/or within a PCSK9 regulatory element, wherein the at least one gene expression regulator comprises a DNA methyltransferase (DNMT), a DNA demethylase, a histone methyltransferase, a histone demethylase or a portion thereof, or a zinc finger protein-based transcription factor or a portion thereof, or a combination thereof, and wherein the at least one DNA binding protein is Cas9, dCas9, Cpf1, a zinc finger nuclease (ZNF), a transcription activator-like effector nuclease (TALEN), a homing endonuclease, a dCas9-FokI nuclease, or a MegaTal nuclease.

In certain embodiments, the PCSK9 regulatory element is a transcription start site, a core promoter, a proximal promoter, a distal enhancer, a silencer, an insulator element, a boundary element, or a locus control region.

In some embodiments, the modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element is located within about 100bp, about 200bp, about 300bp, about 400bp, about 500bp, about 600bp, about 700bp, about 800bp, about 900bp, about 1000bp, about 1100bp, about 1200bp, about 1300bp, about 1400bp or about 1500bp upstream of the transcription start site of the PCSK9 gene. In some embodiments, the modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element is located within 1000bp upstream of the transcription start site of the PCSK9 gene. In some embodiments, the modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element is located within 300bp upstream of the transcription start site of the PCSK9 gene.

In some embodiments, the modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element is located within about 100bp, about 200bp, about 300bp, about 400bp, about 500bp, about 600bp, about 700bp, about 800bp, about 900bp, about 1000bp, about 1100bp, about 1200bp, about 1300bp, about 1400bp or about 1500bp downstream of the transcription start site of the PCSK9 gene. In some embodiments, the modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element is located within about 300bp downstream of the transcription start site of the PCSK9 gene. In some embodiments, the modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element is located within 1000bp upstream of the transcription start site of the PCSK9 gene and within 300bp downstream of the transcription start site.

In some embodiments, the modification of at least one nucleotide is DNA methylation. In some embodiments, the at least one gene expression regulator comprises a DNA methyltransferase (DNMT) or a portion thereof. In some embodiments, the DNA methyltransferase is DNMT3A, DNMT3B, DNMT3L, DNMT1 or DNMT2. In some embodiments, the DNMT3A comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the DNMT3L comprises the amino acid sequence of SEQ ID NO: 24.

In certain embodiments, the at least one gene expression regulator comprises a zinc finger protein-based transcription factor or a portion thereof. In certain embodiments, the zinc finger protein-based transcription factor is a Kruppel-associated repression cassette (KRAB). In certain embodiments, the KRAB comprises the amino acid sequence of SEQ ID NO: 22.

In some embodiments, the at least one gene expression regulator includes a DNA methyltransferase or a portion thereof and a transcription factor or a portion thereof based on a zinc finger protein. In some embodiments, the DNA methyltransferase is selected from DNMT3A and DNMT3L and a combination thereof, and the transcription factor based on the zinc finger protein is KRAB. In some embodiments, the at least one DNA binding protein is Cas9, dCas9, Cpf1, zinc finger nuclease (ZNF), transcription activator-like effector nuclease (TALEN), homing endonuclease, dCas9-FokI nuclease or MegaTal nuclease.

In certain embodiments, the at least one DNA binding protein is dCas9. In certain embodiments, the dCas9 comprises Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, Campylobacter jejuni dCas9, Corynebacterium diphtheria dCas9, Eubacterium ventriosum dCas9, Streptococcus pasteurianus dCas9, Lactobacillus farciminis dCas9, Sphaerochaeta globus dCas9, Azospirillum (e.g., strain B510) dCas9, Gluconacetobacter diazotrophicus dCas9, Neisseria cinerea dCas9, Roseburia enterica dCas9, and Streptococcus pasteurianus dCas9. intestinalis) dCas9, Parvibaculum lavamentivorans) dCas9, Nitratifractor salsuginis (e.g., strain DSM 16511) dCas9, Campylobacter lari (e.g., strain CF89-12) dCas9, Streptococcus thermophilus (e.g., strain LMD-9) dCas9. In some embodiments, the dCas9 comprises the amino acid sequence of SEQ ID NO: 1.

In certain embodiments, the fusion molecule comprises the at least one gene expression regulator fused to the C-terminus, the N-terminus, or both, of the at least one DNA binding protein.

In some embodiments, the at least one gene expression regulator is directly fused to the at least one DNA binding protein. In some embodiments, the at least one gene expression regulator is indirectly fused to the at least one DNA binding protein via a non-regulator, a second regulator, or a linker.

In certain embodiments, the fusion molecule comprises dCas9 fused to KRAB on the C-terminal end and to DNMT3A and DNMT3L on the N-terminal end. In certain embodiments, the fusion molecule comprises the amino acid sequence of SEQ ID NO:97.

In some embodiments, the fusion molecule further comprises at least one nuclear localization sequence. In some embodiments, the at least one nuclear localization sequence is directly fused to the C-terminus, N-terminus, or both of the at least one DNA binding protein. In some embodiments, the at least one nuclear localization sequence is indirectly fused to the C-terminus, N-terminus, or both of the at least one DNA binding protein via a linker.

In some embodiments, the nucleic acid sequence encoding the fusion molecule is deoxyribonucleic acid (DNA). In some embodiments, the nucleic acid sequence encoding the fusion molecule is messenger ribonucleic acid (mRNA).

In certain embodiments, the pharmaceutical composition further comprises at least one single guide RNA (sgRNA) complementary to a DNA sequence near the PCSK9 gene and/or within a PCSK9 regulatory element. In certain embodiments, the sgRNA comprises a nucleic acid sequence of SEQ ID NO: 27-95 or 98-108.

In some embodiments, the fusion molecule is packaged in a liposome or lipid nanoparticle. In some embodiments, the fusion molecule and the sgRNA are packaged in a liposome or lipid nanoparticle. In some embodiments, the fusion molecule and the sgRNA are packaged in the same liposome or lipid nanoparticle. In some embodiments, the fusion molecule and the sgRNA are packaged in different liposomes or lipid nanoparticles.

In certain embodiments, the liposome or the lipid nanoparticle comprises an ionizable lipid (20%-70%, molar ratio), a PEGylated lipid (0%-30%, molar ratio), a supporting lipid (5%-50%, molar ratio) and cholesterol (10%-50%, molar ratio). In certain embodiments, the ionizable lipid is selected from a pH-responsive ionizable lipid, a thermally responsive ionizable lipid and a light-responsive ionizable lipid.

In some embodiments, the fusion molecule is packaged in an AAV vector. In some embodiments, the fusion molecule and the sgRNA are packaged in an AAV vector. In some embodiments, the fusion molecule, the fusion molecule and the sgRNA are packaged in the same AAV vector.

In certain embodiments, the fusion molecule and the sgRNA are packaged in different AAV vectors.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A is a schematic diagram showing the "EPICAS" (also known as "CRISPRoff") dual plasmid system and sgRNA tiled screening design targeting mouse PCSK9 expression. The first plasmid ("catalytic protein" plasmid or "fusion molecule" plasmid) encodes DNMT3A-DNMT3L (3A3L)-dCas9-KRAB under the control of the CAG promoter and a GFP marker separated by a 2A element. The second plasmid ("sgRNA" plasmid) has an sgRNA scaffold under the control of the U6 promoter and an mCherry marker under the control of the CMV promoter. The sgRNA for tiled screening targets +250bp upstream of the transcription start site (TSS) of the mouse PCSK9 protein coding sequence (CDS).

Figure 1B is a bar graph showing relative mRNA expression of mouse AML12 cell line after transfection with catalytic protein plasmid and single PCSK9 sgRNA plasmid. n=3 biological replicates per group.

FIG1C is a bar graph showing relative mRNA expression after transfection of the mouse AML12 cell line with a mixture of catalytic protein plasmids and various PCSK9 sgRNA plasmids.

FIG. 1D is a bar graph showing relative mRNA expression after transfection of mouse Ai9 primary hepatocytes with a mixture of catalytic protein plasmids and various PCSK9 sgRNA plasmids.

Figure 2A is a schematic diagram showing the design of the EPICAS mRNA plasmid. The EPICAS ORF contains a DNMT3A-DNMT3L-dCas9-KRAB expression cassette. The plasmid can be digested at the XbaI and BpiI restriction sites to form a linearized plasmid.

FIG. 2B is an electropherogram of purified mRNA expressed from the EPICAS mRNA plasmid.

FIG. 2C is a schematic diagram showing the Snrpn-GFP reporter system.

FIG. 2D is a series of flow cytometry graphs showing Snrpn-GFP expression 8 days after transfection with Snrpn sgRNA or non-targeting sgRNA or 30 days, 150 days, and 400 days after transfection with Snrpn sgRNA.

FIG. 2E is a line graph showing the percentage of GFP-off cells 8 days after transfection with Snrpn sgRNA or non-targeting sgRNA (NT-sgRNA) or 30 days, 150 days, and 400 days after transfection with SnrpnsgRNA.

Figure 2F is a schematic diagram of DNA methylation levels of the Snrpn locus obtained using bisulfite PCR analysis. Bisulfite sequencing analysis of the Snrpn locus between CRISPRoff targeted and non-targeted cells is shown 8 days after transfection. Each row represents a single clone and sequencing readout, and each column indicates a specific genomic position. White dots represent unmethylated CpG dinucleotides, and black dots represent methylated CpG dinucleotides. The figure below represents methylation at 13 positions of the Snrpn locus.

Figure 2G is a schematic diagram of bisulfite sequencing analysis of the Snrpn locus between CRISPRoff targeted and non-targeted cells 400 days after transfection. The area behind the circle represents the average degree of methylation of each CpG dinucleotide (n=9 sequencing reads).

FIG2H is a capillary gel electrophoresis showing the purity of CRISPRoff mRNA obtained by in vitro transcription.

FIG. 2I is a series of graphs showing the percentage of GFP-off cells at 70 and 90 days after CRISPRoff transfection.

Figure 3A is a schematic diagram of lipid nanoparticle (LNP) design. Epigenetic CRISPR/Cas elements and sgRNA elements can be encapsulated by LNP.

FIG. 3B is a transmission electron microscopy image showing LNPs containing EPICAS.

FIG. 3C is a graph showing the size distribution of LNPs.

FIG. 3D is a series of photographs showing in vivo fluorescence imaging of luciferase mRNA delivered to mouse hepatocytes using lipid nanoparticles via intramuscular injection.

Figure 3E is a schematic diagram of the in vivo experimental design for delivering LNPs containing EPICAS mRNA and mouse PCSK9 targeting sgRNA. LNPs were administered to C57CB/6J mice by injection into the lateral tail vein, and PCSK9 gene expression was analyzed 5 days after injection.

FIG. 3F is a bar graph showing relative mRNA expression of mouse PCSK9 after injection with PBS or LNPs containing EPICAS mRNA and sgRNA targeting mouse PCSK9.

Figure 4A is a schematic diagram of the sgRNA tiling screening design for monkey PCSK9. The sgRNAs in the tiling screening target +250bp upstream of the transcription start site (TSS) of the monkey PCSK9 protein coding sequence (CDS).

FIG4B is a bar graph showing relative mRNA expression following transfection of monkey cells with catalytic protein plasmids and individual PCSK9 sgRNA plasmids.

FIG. 5A is a schematic diagram showing the experimental design of a tiling screen of sgRNAs targeting human PCSK9.

Fig. 5B is a series of bar graphs showing the mean fluorescence intensity rate of PCSK9 after transfection of human PCSK9 reporter cell lines with various sgRNAs targeting the upstream 300bp and downstream 300bp regions of human PCSK9 TSS. The figure on the left shows the results 72 hours after transfection. The figure on the right shows the results 120 hours after transfection.

FIG5C is a series of graphs showing the mean fluorescence intensity of PCSK9 72 hours after transfection of the human PCSK9 reporter cell line with various sgRNAs targeting the 300 bp upstream and 300 bp downstream regions of the human PCSK9 TSS.

Figure 5D is a series of graphs showing PCSK9 mRNA expression after transfection with the EPICAS dual plasmid system using various PCSK9 targeting sgRNAs. The graph on the left shows PCSK9 mRNA expression in a Hep3B cell line endogenously expressing human PCSK9 within 48 hours after transfection with various sgRNAs. The graph on the right shows the mean fluorescence intensity rate of PCSK9 120 hours after transfection of a human PCSK9 reporter cell line with various sgRNAs.

Fig. 6A is an image showing fluorescent staining of mouse liver. TdTomato staining (CRISPRoffmRNA) and DAPI staining are shown. The high proportion of cells showing tdTomato fluorescence shows the efficacy of LNP-mediated CRISPRoffmRNA in mouse liver.

Figure 6B is an image showing mouse organs after LNP-mediated CRISPRoff mRNA delivery. Luciferase imaging showed that CRISPRoff mRNA was localized to the liver.

FIG. 6C is a series of images showing the in vivo distribution of Luc mRNA-LNPs obtained by luciferase imaging at 6 h, 12 h, 24 h, and 48 h after administration.

Fig. 6D is a graph showing the Pcsk9 mRNA levels in wild-type mouse livers. Relative mRNA expression levels were assessed one week after treatment with LNP formulations with CRISPRoff mRNA and Pcsk9 targeting sgRNA at a specified dose (mg RNA/kg body weight). n=5 for PBS and n=6 for LNP formulation groups. mg/kg (MPK).

Figure 6E is a graph showing protein levels of Pcsk9 in the blood of mice treated with LNP formulations with CRISPRoff mRNA and Pcsk9 targeting sgRNA at the indicated doses (mg RNA/kg body weight). n=5 for PBS and n=6 for LNP formulation groups.

Figure 6F is a graph showing the protein level of Pcsk9 in the blood of mice at 2, 4, 6 and 8 weeks after treatment with 3 mg/kg of LNP formulations containing CRISPRoff mRNA and Pcsk9 targeting sgRNA. n=4 for each group.

FIG6G is a schematic diagram of partial hepatectomy (PHx) and liver regeneration experiment.

FIG. 6H is a graph showing comparison of hepatic Pcsk9 mRNA levels in mice 7 days after PHx or sham surgery.

FIG. 6I is a diagram showing targeted bisulfite sequencing analysis of CpG dinucleotides at the promoter of the PCSK9 gene. The methylation level at each CpG dinucleotide is quantified by the average β value from 4 or 3 biological parallel samples. n=4, 3 and 4 for PBS, sham and PHx. PHx, partial hepatectomy. P values are calculated by Student's t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

FIG. 7A is a schematic diagram of Pcsk9 repression by epigenome editing in high-fat diet (HFD) mice.

7B is a graph showing the relative protein levels of Pcsk9 in the blood of HFD mice 7 days after treatment with PBS, 4 mg/kg or 6 mg/kg of LNP formulations containing CRISPRoff mRNA and Pcsk9-targeting sgRNA. mg/kg (MPK).

FIG. 7C is a graph showing the relative protein levels of Pcsk9 in the blood of HFD mice 14 days after treatment.

FIG. 7D is a graph showing comparison of plasma LDL-C levels in HFD mice before and after treatment. n=5 biological replicates per group. P values were calculated by Student's t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig. 8A is a scatter plot of log2-transformed TPMs of genes in two groups. The marked points correspond to the PCSK9 gene. The data represent the average of three independent biological parallel samples.

Figure 8B is a graphical representation of gene expression changes within 1 Mb upstream and downstream of the target gene Pcsk9. The x-axis represents the genomic position of each gene.

Figure 8C is a Manhattan plot showing differentially methylated CpGs across the genome between CRISPRoff and PBS treated mice. -log10 (p-value) greater than 0 indicates a higher methylation level at the locus in CRISPRoff treated mice, -log10 (p-value) less than 0 indicates a higher methylation level in PBS treated mice. Arrows mark the genomic location of Pcsk9.

Figure 8D is a diagram of methylation changes within 1 kb upstream and downstream of Pcsk9. The x-axis represents the genomic position. The y-axis represents -log (p-value). The red dots indicate that the PCSK9 gene is within the range from the transcription start site to the end of its stop codon, and the blue dots indicate that it is outside this range. The protospacer sequence sites targeted by the two sgRNAs are marked as "sgRNA7" and "sgRNA9".

Figure 8E is a volcano plot showing gene expression changes of predicted sgRNA-dependent off-target genes and target genes. The x-axis represents the log2-transformed fold change of the indicated gene obtained by DESeq2, and the y-axis represents the log10-transformed adjusted P value. n = 3 biological replicates per group.

FIG. 9A is a graph showing the size distribution of LNPs encapsulating CRISPRoff and Pcsk9 targeting sgRNAs.

FIG. 9B is a Cryo-EM image of LNPs encapsulating CRISPRoff and Pcsk9 targeting sgRNA.

Figure 9C shows a graph of targeted bisulfite sequencing analysis of CpG dinucleotides on the PCSK9 gene treated with different doses of CRISPRoff-encapsulated LNPs. The methylation level at each CpG dinucleotide was quantified by the average β value from 4 or 3 biological parallel samples. n = 3 per group. mg/kg (MPK).

Figure 9D is a series of graphs showing the absolute values of blood levels of aspartate aminotransferase (ALT), alanine aminotransferase (AST), alkaline phosphatase (ALP), and albumin (ALB) in mice before and 4 and 7 days after treatment with 3 or 6 mg/kg LNP-encapsulated CRISPRoff. n = 6 per group. P values were calculated by Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 10A shows a volcano plot of gene expression changes for the entire transcriptome. Points representing Pcsk9 are indicated.

Figure 10B shows a graph comparing methylation levels within a 50 kb genomic window on either side of the PCSK9 gene. The bars above the x-axis represent loci with methylation levels greater than 0.5, and the bars below the x-axis represent loci with methylation levels less than 0.5. The protospacer sequence sites targeted by the two sgRNAs are labeled "sgRNA7" and "sgRNA9".

FIG. 10C is a bar graph showing the log2 transformed fold changes in methylation and gene expression levels of the indicated genes, demonstrating the statistical significance of gene expression levels between CRISPRoff and PBS treated mice. FIG.

Figure 10D is a bar graph showing methylation of the indicated genes and log2 transformed fold changes in gene expression levels, showing statistical significance of methylation levels between CRISPRoff and PBS treated mice. n=3 biological replicates per group.

Detailed ways

The present disclosure overcomes the problems associated with current technologies by providing genetically engineered fusion molecules (e.g., DNMT3A-DNMT3L (3A3L)-dCas9-KRAB fusion molecules) for targeted reduction or elimination of gene products (e.g., PCSK9) in cells for in vivo gene therapy. The genetically engineered fusion molecules disclosed herein can be used to treat genetic diseases, including, for example, liver diseases, diseases associated with high cholesterol, and diseases associated with cholesterol (e.g., low-density lipoprotein (LDL) cholesterol) disorders. Therefore, methods for preparing genetically engineered fusion molecules and pharmaceutical preparations (e.g., lipid nanoparticle preparations) thereof for in vivo delivery are also provided.

I. Definitions

The term "coding sequence" or "coding nucleic acid" as used herein refers to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence encoding a protein. The coding sequence may further include start and stop signals operably linked to regulatory elements, including a promoter and polyadenylation signal capable of directing expression in cells of an individual or mammal to which the nucleic acid is administered. The coding sequence may be codon optimized.

As used herein, the term "complementary" or "complementary" with respect to nucleic acids can refer to Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of a nucleic acid molecule. "Complementarity" refers to a property shared between two nucleic acid sequences such that when they are aligned antiparallel to each other, the nucleotide bases at each position will be complementary.

The terms "correction", "genome editing" and "restoration" refer to changing a mutant gene that encodes a mutant protein, a truncated protein, or does not encode a protein at all, so as to obtain the expression of a full-length functional or partially full-length functional protein. Correcting or restoring a mutant gene can include replacing a region of the gene with the mutation or replacing the entire mutant gene with a copy of a gene that does not have the mutation using a repair mechanism such as homology-directed repair (HDR). Correcting or restoring a mutant gene can also include repairing a frameshift mutation that causes a premature stop codon, an abnormal splice acceptor site, or an abnormal splice donor site by creating a double-strand break in the gene and then repairing it using non-homologous end joining (NHEJ). NHEJ can add or delete at least one base pair during the repair process, which can restore the correct reading frame and eliminate premature stop codons. Correcting or restoring a mutant gene can also include destroying an abnormal splice acceptor site or a splice donor sequence. Correcting or restoring a mutant gene can also include deleting a non-essential gene segment by the simultaneous action of two nucleases on the same DNA chain, so as to restore the correct reading frame by removing the DNA between the two nuclease target sites and repairing the DNA break by NHEJ.

As used herein, the terms "donor DNA", "donor template" and "repair template" refer to a double-stranded DNA fragment or molecule that includes at least a portion of a gene of interest. The donor DNA may encode a fully functional protein or a partially functional protein.

The term "frameshift" or "frameshift mutation" as used herein is used interchangeably and refers to a type of gene mutation in which the addition or deletion of one or more nucleotides causes the reading frame of the codon in the mRNA to shift. The shift of the reading frame may lead to changes in the amino acid sequence during protein translation, such as missense mutations or premature stop codons.

As used herein, the terms "functional" and "fully functional" describe a protein that has biological activity. A "functional gene" refers to a gene that is transcribed into mRNA, which is translated into a functional protein.

The term "fusion protein" as used herein refers to a chimeric protein produced directly or indirectly by covalent or non-covalent linkage of two or more genes, which originally encoded separate proteins. In certain embodiments, translation of the fusion gene produces a single polypeptide having functional properties derived from each original protein.

The term "genetic construct" as used herein refers to a DNA or RNA molecule comprising a nucleotide sequence encoding a protein. The coding sequence includes start and stop signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in a cell.

The term "homologous directed repair" or "HDR", used interchangeably herein, refers to a mechanism for repairing double-stranded DNA damage in cells when homologous DNA fragments are present in the nucleus (mainly in the G2 and S phases of the cell cycle). HDR uses a donor DNA template to guide repair and can be used to produce specific sequence changes to the genome, including targeted insertion of entire genes. If the donor template is provided together with a site-specific nuclease, such as provided together with a CRISPR/Cas9-based system, the cellular mechanism will repair the break by homologous recombination, which is enhanced by several orders of magnitude in the presence of DNA cutting. When homologous DNA fragments are not present, non-homologous end joining may occur instead.

The term "genome editing" as used herein refers to changing a gene. Genome editing can include correcting or restoring a mutant gene. Genome editing can include knocking out a gene, such as a mutant gene or a normal gene. Genome editing can be used to treat a disease by changing a gene of interest.

In the case of two or more nucleic acid or polypeptide sequences, the term "identical" or "identity" as used herein refers to a sequence having a specific percentage of identical residues within a specified region. The percentage can be calculated as follows: optimally align the two sequences, compare the two sequences within a specified region, determine the number of positions where identical residues occur in the two sequences to produce the number of matching positions, divide the number of matching positions by the total number of positions in the specified region, and multiply the result by 100 to produce the percentage of sequence identity. In the case where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified comparison region includes only a single sequence, the residues of the single sequence are included in the denominator of the calculation, but not in the numerator. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered to be equivalent. Identity can be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0. The identity of related peptides can be easily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al, SIAM J. Applied Math. 48, 1073 (1988), the entireties of which are incorporated herein by reference.

As used herein, the terms "mutant gene" or "mutated gene" used interchangeably herein refer to a gene that has undergone a detectable mutation. A mutant gene has undergone a change, such as a loss, gain or exchange of genetic material, which affects the normal transmission and expression of the gene. As used herein, a "disrupted gene" refers to a mutant gene having a mutation that causes a premature stop codon. The product of a disrupted gene is truncated relative to the full-length non-disrupted gene product.

The term "epigenetic modification regulator" as used herein refers to an agent that targets gene expression by epigenetic modification (e.g., by histone acetylation or methylation, or regulatory elements of target genes such as promoters, enhancers, or DNA methylation at the transcription start site). Chromatin remodeling and DNA methylation are two main mechanisms for regulating gene transcription. Specific epigenetic marks (e.g., DNA methylation) structurally or biochemically guide gene transcription or gene silencing/repression. For example, DNA methylation in a region that regulates transcriptional activity changes gene expression without changing the underlying DNA sequence. Transcriptional regulation using epigenetic modifications (e.g., DNA methylation) allows targeted regulation of gene expression without affecting the expression of other gene products.

The term "non-homologous end joining (NHEJ) pathway" as used herein refers to a pathway for repairing double-strand breaks in DNA by directly joining the broken ends without the need for a homologous template. The template-independent rejoining of DNA ends by NHEJ is a random, error-prone repair process that introduces random micro-insertions and micro-deletions (indels) at the DNA breakpoints. This method can be used to intentionally destroy, delete or change the reading frame of the target gene sequence. NHEJ typically uses short homologous DNA sequences called microhomologs to guide repair. These microhomologs are typically present in single-stranded overhangs at the ends of double-strand breaks. When the overhangs are fully compatible, NHEJ usually accurately repairs the break, however, imprecise repairs that result in nucleotide loss may also occur, but are more common when the overhangs are incompatible.

The term "normal gene" as used herein refers to a gene that has not undergone changes such as loss, gain or exchange of genetic material. A normal gene undergoes normal gene transmission and gene expression.

As used herein, the term "nuclease-mediated NHEJ" refers to NHEJ initiated after a nuclease, such as cas9, cleaves double-stranded DNA.

The term "nucleic acid" or "oligonucleotide" or "polynucleotide" as used herein refers to at least two nucleotides covalently linked together. The description of a single strand also defines the sequence of the complementary strand. Therefore, nucleic acid also encompasses the complementary strand of the described single strand. Many variants of nucleic acid can be used for the same purpose as a given nucleic acid. Therefore, nucleic acid also encompasses substantially identical nucleic acids and their complements. The single strand provides a probe that can hybridize with a target sequence under stringent hybridization conditions. Therefore, nucleic acid also encompasses probes that hybridize under stringent hybridization conditions. Nucleic acid can be single-stranded or double-stranded, or can contain parts having both double-stranded and single-stranded sequences. Nucleic acid can be DNA (both genomic DNA and cDNA), RNA or a hybrid, wherein the nucleic acid can contain a combination of deoxyribonucleotides and ribonucleotides, and a combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine and isoguanine. Nucleic acid can be obtained by chemical synthesis methods or by recombinant methods.

As used herein, the term "operably linked" means that the expression of a gene is under the control of a promoter to which it is spatially linked. A promoter may be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between a promoter and a gene may be about the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variations in this distance may be tolerated without loss of promoter function.

The term "partial functionality" as used herein describes a protein encoded by a mutant gene that has a biological activity lower than a functional protein but higher than a non-functional protein. In one embodiment, a partially functional protein demonstrates a biological activity lower than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% of the biological activity of a corresponding functional protein.

As used herein, the term "premature stop codon" or "out-of-frame stop codon" refers to a nonsense mutation in a DNA sequence that produces a stop codon at a position not normally found in the wild-type gene. A premature stop codon may cause a protein to be truncated or shorter than the full-length version of the protein.

The term "promoter" or "core promoter" as used herein refers to a synthetic or naturally derived molecule that can confer, activate or enhance nucleic acid expression in a cell. A promoter may include one or more specific transcriptional regulatory sequences to further enhance the expression of nucleic acids and/or change the spatial expression and/or temporal expression of nucleic acids. A promoter may also include a distal enhancer or repressor element, which may be located at a position up to several thousand base pairs from the transcription start site. A promoter may be derived from sources including viruses, bacteria, fungi, plants, insects and animals. A promoter may constitutively or relative to cells, tissues or organs in which expression occurs or relative to the developmental stage in which expression occurs or to external stimuli such as physiological stress, pathogens, metal ions or inducers to respond differentially to regulate the expression of gene components. Representative examples of promoters include bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter and CMV IE promoter.

The term "target gene" as used herein refers to any nucleotide sequence encoding a known or putative gene product. The target gene may be a mutant gene involved in a genetic disease or disorder.

As used herein, the term "target region" refers to the region of a target gene to which a site-specific nuclease is designed to bind.

As used herein, the term "transgenic" refers to a gene or genetic material containing a gene sequence that is isolated from one organism and introduced into a different organism. Alternatively, the term "transgenic" also refers to a gene or genetic material that is chemically synthesized and introduced into an organism. This non-natural DNA segment can retain the ability to produce RNA or protein in a transgenic organism, or it can change the normal function of the genetic code of a transgenic organism. The introduction of a transgenic has the potential to change the phenotype of an organism.

As used herein, the term "variant" refers to (i) a portion or fragment of a reference nucleotide sequence; (ii) a complementary sequence of a reference nucleotide sequence or a portion thereof; (iii) a nucleic acid substantially identical to a reference nucleic acid or its complementary sequence; or (iv) a nucleic acid that hybridizes to a reference nucleic acid, its complementary sequence, or a sequence substantially identical thereto under stringent conditions. For peptides or polypeptides, the amino acid sequence of a "variant" differs by insertion, deletion, or conservative substitution of amino acids, but retains at least one biological activity.

Variant can also refer to a protein having an amino acid sequence substantially identical to the amino acid sequence of a reference protein retaining at least one biological activity. Conservative substitution of amino acids, i.e. replacing amino acids with different amino acids having similar properties (e.g., hydrophilicity, degree and distribution of charged regions), is considered in the art to generally involve minor changes. As understood in the art, these minor changes can be identified in part by considering the hydropathic index of amino acids. Kyte et al., J. Mol. Biol. 157: 105-132 (1982), which is incorporated herein by reference in its entirety. The hydropathic index of amino acids is a consideration based on their hydrophobicity and charge. It is known in the art that amino acids with similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids with a hydropathic index of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that will produce proteins that retain biological function. Considering the hydrophilicity of amino acids in the case of peptides allows the calculation of the maximum local average hydrophilicity of the peptide. Amino acids with hydrophilicity values within ±2 of each other can be substituted. Both the hydrophobicity index and the hydrophilicity value of an amino acid are influenced by the particular side chain of that amino acid. Consistent with this observation, amino acid substitutions compatible with biological function are understood to depend on the relative similarity of the amino acids, particularly those of the side chains, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

When used in this article, the term "vector" used herein refers to a nucleic acid sequence containing a replication origin. The vector can be a viral vector, a bacteriophage, a bacterial artificial chromosome or a yeast artificial chromosome. The vector can be a DNA or RNA vector. The vector can be a self-replicating extrachromosomal vector, such as a DNA plasmid.

As used herein, the terms "gene transfer," "gene delivery," and "gene transduction" refer to methods or systems for reliably inserting specific nucleotide sequences (such as DNA or RNA), fusion proteins, polypeptides, etc. into target cells.

As used herein, the terms "adeno-associated virus (AAV) vector", "AAV gene therapy vector" and "gene therapy vector" refer to vectors having functional or partially functional ITR sequences and transgenes. As used herein, the term "ITR" refers to an inverted terminal repeat (ITR). The ITR sequence can be derived from an adeno-associated virus serotype, including but not limited to AAV-1, AAV-2, AAV-3, AAV-4, AAV-5 and AAV-6. However, the ITR does not have to be a wild-type nucleotide sequence and can be altered (e.g., by insertion, deletion or substitution of nucleotides) as long as the sequence retains function to provide functional rescue, replication and packaging. One or more AAV wild-type genes of the AAV vector, preferably the rep and/or cap genes, can be deleted in whole or in part, but retain functional flanking ITR sequences. The function of the functional ITR sequence is, for example, to rescue, replicate and package AAV virus particles or particles. Therefore, an "AAV vector" is defined herein as including at least those sequences required for inserting a transgene into a subject's cells. Optionally, sequences that are necessary to be in cis for viral replication and packaging (e.g., functional ITRs) are included.

As used herein, the term "gene therapy" refers to a method of treating a patient in which a polypeptide or nucleic acid sequence is transferred into the patient's cells in order to modulate the activity and/or expression of a particular gene. In some embodiments, the expression of the gene is inhibited. In some embodiments, the expression of the gene is enhanced. In some embodiments, the temporal or spatial pattern of gene expression is modulated.

The "transgene" may contain a transgenic sequence or a native or wild-type DNA sequence. The transgenic may become part of the genome of a primate subject. The transgenic sequence may be partially or completely species heterologous, i.e., the transgenic sequence or a portion thereof may be from a species different from the cell into which it is introduced.

As used herein, the term "stably maintained" refers to a feature of a transgenic subject (e.g., a human or non-human primate) that maintains at least one transgenic element (i.e., a desired element) through multiple generations of cells. For example, the term is intended to encompass many cell division cycles of the initially transfected cells. The term "stable transfection" or "stably transfected" refers to the introduction and integration of exogenous DNA into the genome of a cell. The term "stable transfectant" refers to a cell that has stably integrated exogenous DNA into genomic DNA.

As used herein, the terms "transgene encoding ...," "nucleic acid molecule encoding ...," "DNA sequence encoding ...," and "DNA encoding ..." refer to the order or sequence of deoxyribonucleotides along a deoxyribonucleic acid chain. For example, the order of these deoxyribonucleotides can determine the order of amino acids along a polypeptide (protein) chain. Thus, the DNA sequence can encode the amino acid sequence.

The term "wild type" (wt) as used herein refers to a gene or gene product having the characteristics of the gene or gene product when isolated from a naturally occurring source. A wild-type gene is a gene most often observed in a population and is therefore arbitrarily designed to be the "normal" or "wild-type" form of a gene. In contrast, the term "modified" or "mutant" refers to a gene or gene product that exhibits modification (i.e., the characteristics of a change) in terms of sequence and/or functional properties when compared to a wild-type gene or gene product. It is noteworthy that naturally occurring mutants can be isolated that are identified by obtaining the characteristics of a change compared to a wild-type gene or gene product.

The term "transfection" as used herein refers to the uptake of foreign nucleic acids (e.g., DNA or RNA) by a cell. A cell has been "transfected" when an exogenous nucleic acid (DNA or RNA) is introduced into the interior of a cell membrane. Many transfection techniques are well known in the art (see, e.g., Graham et al., Virol., 52: 456 (1973); Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratories, New York (1989); Davis et al., Basic Methods in Molecular Biology, Elsevier, (1986); and Chu et al., Gene 13: 197 (1981), which are incorporated herein by reference in their entirety). Such techniques can be used to introduce one or more exogenous DNA portions, such as gene transfer vectors and other nucleic acid molecules, into suitable recipient cells.

As used herein, the terms "stable transfection" and "stably transfected" refer to the introduction and integration of foreign DNA into the genome of the transfected cell. The term "stable transfectant" refers to a cell that has stably integrated foreign DNA into its genomic DNA.

As used herein, the term "transient transfection" or "transiently transfected" refers to the introduction of foreign DNA into a cell, wherein the foreign DNA fails to integrate into the genome of the transfected cell and is maintained as an episome. During this period, the foreign DNA is regulated by the expression of endogenous genes in the chromosome. The term "transient transfectant" refers to a cell that has taken up foreign DNA but has failed to integrate the DNA. As used herein, the term "transduction" means delivering a DNA molecule to a recipient cell in vivo or in vitro by a replication-defective viral vector, such as by a recombinant AAV viral particle.

The term "recipient cell" as used herein refers to a cell that has been transfected or transduced, or is capable of being transfected or transduced, by a nucleic acid construct or vector carrying a selected nucleotide sequence of interest. The term includes progeny of a parent cell, whether or not the progeny is identical to the original parent in morphology or genetic makeup, as long as the selected nucleotide sequence is present. A recipient cell can be a cell of a subject to which the gene therapy particles and/or gene therapy vector have been administered.

As used herein, the term "recombinant DNA molecule" refers to a DNA molecule composed of DNA fragments linked together by molecular biology techniques.

As used herein, the term "regulatory element" refers to a genetic element that can control the expression of a nucleic acid sequence. For example, a promoter is a regulatory element that helps initiate transcription of an operably linked coding region. Other regulatory elements are splicing signals, polyadenylation signals, termination signals, etc.

The term DNA "control sequences" refers collectively to regulatory elements such as promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, replication origins, internal ribosome entry sites ("IRES"), enhancers, etc., which together provide for the replication, transcription, and translation of coding sequences in recipient cells. Not all of these control sequences need to be present.

The term "enhancer" as used herein refers to a non-coding DNA sequence comprising multiple activators and repressor binding sites. The length range of the enhancer is 50bp to 1500bp, and it can be located at the proximal end 5' upstream of the promoter, in any intron of the regulated gene, or at the distal end, in the intron of the adjacent gene, or in the intergenic region away from the locus, or in the region on different chromosomes. More than one enhancer can interact with the promoter. Similarly, the enhancer can regulate more than one gene without linkage restriction, and can "skip" adjacent genes to regulate more distant genes. Transcriptional regulation may involve elements located on chromosomes different from the chromosomes where the promoter is located. The proximal enhancer or promoter of the adjacent gene can serve as a platform for recruiting more distal elements. The enhancer and promoter used can be "endogenous", "exogenous" or "heterologous" relative to the genes to which they are operably connected. "Endogenous" enhancer/promoter is an enhancer/promoter naturally associated with a given gene in the genome. A "foreign" or "heterologous" enhancer or promoter is one that has been placed in juxtaposition to a gene by genetic manipulation (ie, molecular biology techniques) such that transcription of the gene is directed by the ligated enhancer/promoter.

The term "insulator" or "insulator element" as used herein refers to a genetic boundary element that blocks the interaction between an enhancer and a promoter. By being located between an enhancer and a promoter, an insulator can inhibit their subsequent interaction. An insulator can determine the genome that an enhancer can affect. When two adjacent genes on a chromosome have very different transcription patterns and the induction or inhibition mechanism of one of the genes does not interfere with the adjacent gene, an insulator is needed. It has also been found that insulators are gathered at the boundaries of topologically associated domains (TADs) and may play a role in dividing the genome into "chromosome neighborhoods", that is, the genomic regions where regulation occurs. Insulator activity is believed to be mainly achieved by the 3D structure of DNA mediated by proteins including CTCF. Insulators may play a role through a variety of mechanisms. Many enhancers form DNA loops that physically approach the promoter region during transcriptional activation. Insulators may promote the formation of DNA loops, thereby preventing the formation of promoter-enhancer loops. Barrier insulators can prevent heterochromatin from spreading from silent genes to actively transcribed genes.

The term "locus control region" as used herein refers to a long-distance cis-regulatory element that can enhance the expression of a gene connected to a distal chromatin site. It functions in a copy number-dependent manner and has tissue specificity, such as the selective expression of 3-globulin genes in erythrocytes. The expression level of a gene can be changed by LCR and gene proximal elements (e.g., promoters, enhancers, and silencers). LCR functions by recruiting chromatin modifications, coactivators, and transcriptional complexes. Its sequence is conserved in many vertebrates, and the conservation of specific sites may indicate the importance of its function.

The terms "silencer" or "repressor" as used herein are used interchangeably and refer to DNA sequences that can bind transcriptional regulators and prevent genes from being expressed as proteins. A silencer is a sequence-specific element that can negatively affect the transcription of its specific gene. Silencer elements can be located at many locations in DNA. The most common location is upstream of the target gene, where it can help inhibit the transcription of the gene. The distance may vary greatly between about -20bp and -2000bp upstream of the gene. Some silencers are located downstream of the promoter within the introns or exons of the gene itself. Silencers are also found in the 3' untranslated region (3'UTR) of mRNA. There are two main types of silencers in DNA, namely classical silencer elements and non-classical negative regulatory elements (NREs). In classical silencers, genes are actively inhibited by silencer elements, mainly by interfering with the assembly of general transcription factors (GTFs). NREs passively inhibit genes, usually by inhibiting other elements upstream of the gene.

As used herein, the term "tissue specificity" refers to regulatory elements or control sequences such as promoters, enhancers, etc., wherein the expression of a nucleic acid sequence is significantly higher in a specific cell type or tissue.

The presence of a "splicing signal" on an expression vector generally results in a higher expression level of the recombinant transcript. The splicing signal mediates the removal of introns from the primary RNA transcript and consists of a splicing donor and acceptor site (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York (1989), pp. 16.7-16.8, which is incorporated herein by reference in its entirety). Commonly used splicing donor and acceptor sites are the splicing junctions of the 16S RNA from SV40.

Transcription termination signals are usually present downstream of the polyadenylation signal and are several hundred nucleotides in length. The term "poly A site" or "poly A sequence" as used herein refers to a DNA sequence that directs both termination and polyadenylation of a nascent RNA transcript. Effective polyadenylation of a recombinant transcript is desirable because transcripts lacking a poly A tail are unstable and rapidly degraded. The poly A signal used in the expression vector may be "heterologous" or "endogenous". An endogenous poly A signal is a signal naturally present at the 3' end of the coding region of a given gene in the genome. A heterologous poly A signal is a signal that is separated from a gene and operably connected to the 3' end of another gene. A commonly used heterologous poly A signal is the SV40 poly A signal. The SV40 poly A signal is contained on a 237bp BamHI/BclI restriction fragment and directs both termination and polyadenylation (Sambrook et al., supra, 16.6-16.7, which are incorporated herein by reference in their entirety).

The terms "subject" and "patient" as used herein are used interchangeably herein and refer to humans and non-human animals. The term "non-human animal" of the present disclosure includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.

As defined herein, a "therapeutically effective amount" or "therapeutically effective dose" is an amount or dose of a fusion protein, polypeptide, nucleic acid, lipid nanoparticle, liposome, AAV particle, or viral particle that is capable of producing a sufficient amount of the desired protein to modulate the activity of the protein in the desired manner, thereby providing a relief tool for clinical intervention. In certain embodiments, the therapeutically effective amount or dose of the transfected fusion protein, polypeptide, nucleic acid, AAV particle, or viral particle as described herein is sufficient to inhibit the gene targeted by the fusion protein/gene therapy construct.

As used herein, the term "treating," e.g., treating, a disease, refers to a subject (e.g., a human) who has a disease, is at risk of having a disease, and/or experiences symptoms of a disease, and in one embodiment, when administered, e.g., a fusion molecule or a nucleic acid encoding the fusion molecule and/or a gRNA or a nucleic acid encoding the gRNA as described herein, will suffer milder symptoms and/or will recover faster than if the fusion molecule or a nucleic acid encoding the fusion molecule and/or the gRNA or a nucleic acid encoding the gRNA had never been administered.

II. DNA Binding Proteins

In certain embodiments of the methods and compositions as defined herein according to the present disclosure, the DNA binding protein (e.g., DNA targeting agent) includes a (DNA) nuclease, such as a nuclease that can target DNA in a sequence-specific manner or can direct or indicate targeting DNA in a sequence-specific manner, such as a CRISPR-Cas system, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN) or a meganuclease. In certain embodiments, the DNA binding protein is a DNA nuclease derived from a CRISPR-Cas system.

Transcription Activator-Like Effector Nuclease (TALEN) System

In certain embodiments, the nucleic acid binding protein is a (modified) transcription activator-like effector nuclease (TALEN) system. Transcription activator-like effectors (TALEs) can be engineered to bind to virtually any desired DNA sequence. Exemplary methods for genome editing using the TALEN system can be found, for example, in the following literature: Cermak T. Doyle EL. Christian M. Wang L. Zhang Y. Schmidt C, et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 2011; 39: e82; Zhang F. Cong L. Lodato S. Kosuri S. Church GM. Arlotta PE Efficient construction of sequence-specific TALeffectors for modulating mammalian transcription. Nat Biotechnol. 2011; 29: 149-153, and U.S. Patent Nos. 8,450,471, 8,440,431 and 8,440,432, each of which is incorporated herein by reference in its entirety.

As further guidance, but not limited thereto, naturally occurring TALEs or "wild-type TALEs" are nucleic acid binding proteins secreted by a variety of Proteobacterial species. TALE polypeptides contain a nucleic acid binding domain composed of tandem repeats of highly conserved monomeric polypeptides that are primarily 33, 34, or 35 amino acids in length, differing from each other primarily at amino acid positions 12 and 13. In certain embodiments, the nucleic acid is DNA.

As used herein, the term "polypeptide monomer" or "TALE monomer" is used to refer to the highly conserved repetitive polypeptide sequence within the TALE nucleic acid binding domain, and the term "repeat variable diresidue" or "RVD" is used to indicate the highly variable amino acids at positions 12 and 13 of the polypeptide monomer.

As provided throughout this disclosure, the amino acid residues of the RVD are described using the IUPAC single-letter code for amino acids. The general representation of the TALE monomer contained in the DNA binding domain is X1-11-(X12X13)-X14-33 or 34 or 35, wherein the subscript represents the amino acid position and X represents any amino acid. X12X13 represents the RVD. In some polypeptide monomers, the variable amino acid at position 13 is missing or absent, and in such polypeptide monomers, the RVD consists of a single amino acid. In such cases, the RVD can be alternatively represented as X*, wherein X represents X12, and (*) represents that X13 does not exist. The DNA binding domain comprises several repeats of the TALE monomer, which can be represented as (X1-11-(X12X13)-X14-33 or 34 or 35)z, wherein in a favorable embodiment, z is at least 5 to 40. In another favorable embodiment, z is at least 10 to 26. TALE monomers have nucleotide binding affinity, which is determined by the identity of the amino acids in the RVD. For example, a polypeptide monomer with an RVD of NI preferentially binds to adenine (A), a polypeptide monomer with an RVD of NG preferentially binds to thymine (T), a polypeptide monomer with an RVD of HD preferentially binds to cytosine (C), and a polypeptide monomer with an RVD of NN preferentially binds to both adenine (A) and guanine (G). In another embodiment of the present disclosure, a polypeptide monomer with an RVD of IG preferentially binds to T. Therefore, the number and order of polypeptide monomer repetitions in the nucleic acid binding domain of TALE determine its nucleic acid target specificity. In a further embodiment of the present disclosure, a polypeptide monomer with an RVD of NS recognizes all four base pairs and can bind to A, T, G or C. The structure and function of TALEs are further described in, for example, Moscou et al., Science 326:1501 (2009); Boch et al., Science 326:1509-1512 (2009); and Zhang et al., Nature Biotechnology 29:149-153 (2011), each of which is incorporated herein by reference in its entirety. In certain embodiments, targeting is achieved by TALEN fragments that bind to polynucleic acids. In certain embodiments, the targeting domain comprises or consists of a catalytically inactive TALEN or a nucleic acid binding fragment thereof.

Zinc Finger Nuclease (ZFN) System

In certain embodiments, the nucleic acid structural protein (e.g., DNA binding protein) comprises or consists of a (modified) zinc finger nuclease (ZFN) system. The ZFN system uses an artificial restriction enzyme generated by fusing a zinc finger DNA binding domain to a DNA cleavage domain, which can be engineered to target a desired DNA sequence. Exemplary methods of genome editing using ZFNs can be found in, for example, U.S. Patent Nos. 6,534,261, 6,607,882, 6,746,838, 6,794,136, 6,824,978, 6,866,997, 6,933,113, 6,979,539, 7,013,219, 7,030,215, 7,220,719, 7,241,573, 7,241,574, 7,585,849, 7,595,376, 6,903,185, and 6,479,626, each of which is incorporated herein by reference in its entirety. As further guidance, but not limited thereto, artificial zinc finger (ZF) technology involves arrays of ZF modules to target new DNA binding sites in the genome. Each finger module in the ZF array targets three DNA bases. Custom arrays of individual zinc finger domains are assembled into ZF proteins (ZFPs). ZFPs can include functional domains. The first synthetic zinc finger nuclease (ZFN) was developed by fusing the ZF protein with the catalytic domain of the IIS type restriction enzyme FokI. (Kim, Y.G. et al., 1994, Chimeric restriction endonuclease, Proc. Natl. Acad. Sci. U.S.A. 91, 883-887; Kim, Y.G. et al., 1996, Hybrid restriction enzymes: zinc finger fusions to FokI cleavage domain. Proc. Natl. Acad. Sci. U.S.A. 93, 1156-1160). By using paired ZFN heterodimers, each of which targets a different nucleotide sequence separated by a short spacer, improved cutting specificity can be obtained while reducing off-target activity. (Doyon, Y.et al., 2011, Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures.Nat.Methods 8, 74-79). ZFP can also be designed as transcriptional activators and repressors, and has been used to target many genes in a variety of organisms. In some embodiments, the targeting domain comprises or consists of a zinc finger nuclease or a nucleic acid binding fragment thereof that binds nucleic acid. In some embodiments, the zinc finger nuclease (its fragment) that binds nucleic acid is catalytically inactive.

Meganuclease

In some embodiments, the nucleic acid structural protein (e.g., DNA binding protein) comprises a (modified) meganuclease, which is an endodeoxyribonuclease characterized by a large recognition site (a double-stranded DNA sequence of 12-40 base pairs). Exemplary methods for using meganucleases can be found in U.S. Patent Nos. 8,163,514, 8,133,697, 8,021,867, 8,119,361, 8,119,381, 8,124,369, and 8,129,134, each of which is incorporated herein by reference in its entirety. In some embodiments, targeting is achieved by a meganuclease fragment that binds to polynucleic acids. In some embodiments, targeting is achieved by a catalytically inactive meganuclease (fragment) that binds to polynucleic acids. Therefore, in a specific embodiment, the targeting domain comprises a meganuclease or a nucleic acid binding fragment thereof that binds to nucleic acids, or is composed of it.

CRISPR-Cas system

In certain embodiments, the nucleic acid structural protein (e.g., DNA binding protein) and the single guide RNA sequence are derived from the CRISPR-Cas system. The present disclosure provides an engineered system based on CRISPR/Cas9 for genome editing and treatment of genetic diseases. The engineered system based on CRISPR/Cas9 can be designed to target any gene (e.g., PCSK9), including genes related to genetic diseases, liver diseases, and cholesterol, such as LDL disorders. The present disclosure provides a CRISPR-Cas system comprising a genetically engineered Cas protein and/or guide RNA with the desired specificity and activity (e.g., reducing or eliminating the expression of the PCSK9 gene product). The CRISPR/Cas9-based system may include a Cas9 protein, a mutated Cas9 protein, or a Cas9 fusion protein (e.g., DNMT3A-DNMT3L (3A3L)-dCas9-KRAB fusion molecule) and at least one sgRNA (e.g., PCSK9 sgRNA). The Cas9 fusion protein may, for example, include a domain having different activities from the endogenous domain of Cas9 (e.g., DNMT3A, DNMT3L, or KRAB).

In general, Cas proteins (which can be used interchangeably herein with CRISPR proteins, CRISPR enzymes, CRISPR-Cas proteins, CRISPR-Cas enzymes, Cas, CRISPR effectors, or Cas effector proteins) and/or guide sequences are components of CRISPR-Cas systems. CRISPR-Cas systems or CRISPR systems collectively refer to transcripts and other elements that participate in the expression of CRISPR-associated ("Cas") genes or direct their activity, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr-mate sequences (covering "direct repeats" and tracrRNA-processed partial direct repeats in the case of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the case of endogenous CRISPR systems), or the term "RNA" used herein (e.g., RNA, such as CRISPR RNA and trans-activating (tracr) RNA or single guide RNA (also referred to as sgRNA; chimeric RNA)) or other sequences and transcripts from CRISPR loci.

In general, the CRISPR system is characterized by an element that promotes the formation of the CRISPR complex at the target sequence site (also referred to as a pre-spacer sequence in the case of an endogenous CRISPR system). In the engineered system of the present disclosure, the direct repeat sequence (direct repeat) may include a naturally occurring sequence or a non-naturally occurring sequence. The direct repeat sequence of the present disclosure is not limited to the length and sequence of naturally occurring. In addition, the direct repeat sequence of the present disclosure may include the insertion of nucleotides, such as an aptamer or a sequence that binds to an adapter protein (for binding to a functional domain). In certain embodiments, one end of the direct repeat sequence containing, for example, an insertion is roughly the first half of a short DR, and the end is roughly the second half of the short DR.

In the context of forming a CRISPR complex, a "target sequence" or "target polynucleotide" refers to a sequence to which a guide sequence is designed to have complementarity, wherein hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. The target sequence may include any polynucleotide, such as a DNA or RNA polynucleotide. In certain embodiments, the target sequence is located in the nucleus or cytoplasm of a cell.

Typically, a guide sequence (or spacer sequence) can be any polynucleotide sequence that has sufficient complementarity to a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. In certain embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is equal to or greater than about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more when optimally aligned using a suitable alignment algorithm.

In certain embodiments, modulation of cleavage efficiency can be utilized by introducing mismatches, such as 1 or more mismatches, such as 1 or 2 mismatches between the spacer sequence and the target sequence (including the position of the mismatches along the spacer/target). For example, the closer the double mismatch is to the center (i.e., not at 3' or 5'), the greater the effect on cleavage efficiency. Therefore, by selecting the position of the mismatch along the spacer, the cleavage efficiency can be modulated. For example, if less than 100% cleavage of the target is desired (e.g., in a cell population), 1 or more, such as preferably 2, mismatches between the spacer and the target sequence can be introduced into the spacer sequence. The closer the position of the mismatch is to the center along the spacer, the lower the percentage of cleavage.

The CRISPR-Cas system or its components can be used to introduce one or more mutations in a target locus or nucleic acid sequence. The mutation can include introducing, deleting or replacing one or more nucleotides at each target sequence of the cell by a guide RNA or sgRNA. The mutation can include introducing, deleting or replacing 1-75 nucleotides at each target sequence of the cell by a guide RNA.

Typically, in the case of an endogenous CRISPR-Cas system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage in or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs of) the target sequence, but may depend, for example, on secondary structure, particularly in the case of RNA targets. In some cases, in the case of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands (if applicable) in or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs of) the target sequence.

In certain embodiments, the guide RNA (capable of guiding Cas to the target locus) may comprise (1) a guide sequence capable of hybridizing to a target locus (a polynucleotide target locus, such as an RNA target locus) in a eukaryotic cell; (2) a direct repeat (DR) sequence present in a single RNA, i.e., sgRNA (arranged in a 5' to 3' direction) or crRNA.

For general information about CRISPR-Cas systems, components thereof, and delivery of such components, including all methods, materials, delivery vehicles, vectors, particles, AAVs, and preparation and use thereof, including amounts and formulations, useful in the practice of the present disclosure, reference is made to the following: U.S. Patent Nos. 8,999,641; 8,993,233; 8,945,839; 8,932,814; 8,906,616; 8,895,308; 8,889,418; 8,889,356; 8,871,445; 8,865,406; 8,795,965; 8,771,945; and 8,697,359; U.S. Patent Publication Nos. US 2014-0310830; US 2014-0287938 A1; US 2014-0273234; A1, US2014-0273232 A1, US 2014-0273231 A1, US2014-0256046 A1, US 2014-0248702 A1, US2014-0242700 A1, US 2014-0242699 A1, US 2014-0242664 A1, US 20 14-0234972 A1, US2014-0227787 A1, US 2014-0189896 A1, US 2014-0186958, US 2014-0186919 A1, US2014-0186843 A1, US 2014-0179770 A1, US 2014-0179006 A1, US 2014-0170753; European patents EP 2784162 B1 and EP 2771468 B1; European patent applications EP 2771468, EP 2764103 and EP 2784162; and PCT patent publications WO 2021/183807A1 (PCT/US2021/021973), WO 2014/093661 (PCT/US2013/074743), WO 2014/093694 (PCT/US2013/074790), WO 2014/093595 (PCT/US2013/074611), WO 2014/093718(PCT/US2013/074825), WO 2014/093709(PCT/US2013/074812), WO 2014/093622(PCT/US2013/074667), WO 2014/093635(PCT/US2013/074691), WO 2014/093655(PCT/US2013/074736), WO 2014/093712(PCT/US2013/074819), WO 2014/093701(PCT/US2013/074800), WO 2014/018423(PCT/US2013/051418), WO 2014/204723 (PCT/US2014/041790), WO 2014/204724 (PCT/US2014/041800), WO 2014/204725 (PCT/US2014/041803), WO 2014/204726 (PCT/US2014/041804), WO 2014/204727 (PCT/US2014/041806), WO 2014/204728 (PCT/US2014/041808), and WO 2014/204729 (PCT/US2014/041809), each of which is incorporated herein by reference in its entirety.

Cas proteins

The Cas protein (e.g., engineered Cas protein) may have a nuclease activity substantially the same as the corresponding wild-type Cas protein (e.g., between 80% and 100%, between 90% and 100%, between 95% and 100%, between 98% and 100%, between 99% and 100%, between 99.9% and 100%, or about 100%). In some cases, the engineered Cas protein has a higher nuclease activity than the corresponding wild-type Cas protein (e.g., at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%).

Alternatively or in addition, the Cas protein (e.g., engineered Cas protein) may have a specificity that is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% higher than the corresponding wild-type Cas protein. In a particular example, the Cas protein (e.g., engineered Cas protein) may have a specificity that is at least 30% higher than the corresponding wild-type Cas protein. The term "specificity" of Cas as used herein may correspond to the number or percentage of on-target polynucleotide cleavage events relative to the number or percentage of all polynucleotide cleavage events including on-target and off-target events. The activity and specificity of the Cas protein are consistent with those described in the following literature: Hsu PD et al., DNA targeting specificity of RNA-guided Cas9 nucleases, Nat Biotechnol. 2013 Sep; 31 (9): 827-832; and Slaymaker IM, et al., Rationally engineered Cas9 nucleases with improved specificity, Science. 2016 Jan l; 351 (6268): 84-88, which also describe examples of methods for detecting the activity and specificity of Cas proteins, and are incorporated herein by reference in their entirety and described in detail elsewhere herein.

In some embodiments, the Cas protein (e.g., its RuvC domain) can slide one base upstream (relative to PAM) and produce staggered cuts, which can be filled and cause the duplication of a single base (i.e., +1 insertion). Examples of +1 insertion positions are described in Zuo, Z., and Liu, J. (2016). Cas9-catalyzed DNA Cleavage Generates Staggered Ends: Evidence from Molecular Dynamics Simulations. Scientific Reports 6, 37584. In some embodiments, the engineered Cas protein has a +1 insertion frequency different from that of the corresponding wild-type Cas protein. For example, when guanine is present in the -2 position relative to PAM, the +1 insertion frequency is higher than the +1 insertion frequency when thymidine, cytidine or adenine is present in the -2 position relative to PAM. In some cases, +1 insertion depends on the host mechanism in human cells. In some instances, the Cas protein can produce staggered cuts. The staggered cuts can be 1 bp or 1 nucleotide 5' overhanging part. The staggered cuts may be 1 bp or 1 nucleotide 3' overhangs.

The nucleic acid molecule encoding Cas can be codon optimized. In this case, an example of a codon optimized sequence is a sequence optimized for expression in a eukaryote, such as humans (i.e., a sequence optimized for expression in humans), or a sequence optimized for expression in another eukaryote, animal, or mammal discussed herein; see, for example, WO 2014/093622 (PCT/US2013/074667) SaCas9 human codon optimized sequence. Although this is preferred, it should be understood that other examples are also possible, and codon optimization for host species other than humans or codon optimization for specific organs is known. In certain embodiments, the enzyme coding sequence encoding Cas is codon optimized for expression in a specific cell, such as a eukaryotic cell. The eukaryotic cell may be a cell of a specific organism or may be derived from a specific organism, such as a mammal, including but not limited to humans or non-human eukaryotes or animals or mammals discussed herein, such as mice, rats, rabbits, dogs, livestock, or non-human mammals or primates. In certain embodiments, processes for modifying the germline genetic characteristics of humans and/or processes for modifying the genetic characteristics of animals, and animals produced by such processes, which may cause humans or animals to suffer without any substantial medical benefit to them, can be excluded. Generally, codon optimization refers to the process of modifying nucleic acid sequences to enhance expression in host cells of interest by replacing at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) of a native sequence with a codon that is more frequently or most frequently used in the genes of a host cell and simultaneously maintaining a native amino acid sequence. Various species show specific preferences for certain codons of specific amino acids. Codon bias (differences in codon usage between organisms) is generally related to the translation efficiency of messenger RNA (mRNA), and the translation efficiency is then believed to depend on the properties of the codons translated and the availability of specific transfer RNA (tRNA) molecules, etc. The quantitative advantage of selected tRNA in a cell generally reflects the most frequently used codons in peptide synthesis. Therefore, genes can be customized based on codon optimization for optimal gene expression in a given organism. Codon usage tables are readily available, for example, in the "Codon Usage Database" at www.kazusa.orjp/codon/, and these tables can be adjusted in a variety of ways. See Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28: 292 (2000). Computer algorithms for codon optimization of specific sequences for expression in specific host cells are also available, such as Gene Forge (Aptagen; Jacobus, PA). In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more or all codons) in the sequence encoding Cas correspond to the most frequently used codons for a specific amino acid.

In some embodiments, the Cas protein may have nucleic acid cleavage activity. The Cas protein may have RNA binding and DNA cutting functions. In some embodiments, Cas may be at a position in or near a target sequence, such as within a target sequence and/or within the complement of a target sequence or at a sequence associated with a target sequence, such as within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500 or more base pairs from the first or last nucleotide of the target sequence, to guide the cutting of one or two nucleic acid chains. In some embodiments, the Cas protein may be within the target sequence and/or within the complement of a target sequence or at a sequence associated with a target sequence and/or within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500 or more base pairs from the first or last nucleotide of the target sequence, to guide more than one cutting (e.g., 1, 2, 3, 4, 5 or more cuttings) of one or two chains. In certain embodiments, the cleavage may be blunt-ended, i.e., produce blunt ends. In certain embodiments, the cleavage may be staggered, i.e., produce sticky ends.

In certain embodiments, the vector encodes a nucleic acid-targeting Cas protein that can be mutated relative to the corresponding wild-type enzyme, so that the mutant nucleic acid-targeting Cas protein lacks the ability to cut one or both chains of a target polynucleotide containing a target sequence, such as a change or mutation in the HNH domain, to produce a mutant Cas that substantially lacks all DNA cleavage activity, such as a mutant enzyme whose DNA cleavage activity is about no more than 25%, 10%, 5%, 1%, 0.1%, 0.01% or less of the nucleic acid cleavage activity of the non-mutated form of the enzyme; an example can be that the nucleic acid cleavage activity of the mutant form is zero or negligible compared to the non-mutated form. For enzymes, the term "derivative" as used herein means that the derivative enzyme is largely based on the wild-type enzyme (in the sense of having a high degree of sequence homology with the wild-type enzyme), but it has been mutated (modified) in a manner known in the art or described herein.

Typically, in the case of an endogenous nucleic acid targeting system, the formation of a nucleic acid targeting complex (comprising a guide RNA or crRNA hybridized to a target sequence and complexed with one or more nucleic acid targeting effector proteins) results in the cleavage of a DNA strand in or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs of) the target sequence. As used herein, the term "sequence associated with a target locus of interest" refers to a sequence near a target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs from the target sequence, wherein the target sequence is contained within the target locus of interest).

It should be understood that the effector protein is based on or derived from an enzyme, so in certain embodiments the term "effector protein" certainly includes "enzyme". However, it should also be understood that in certain embodiments, the effector protein may have DNA or RNA binding activity, but not necessarily cleavage or nicking activity, including dead Cas protein function, as desired.

In some embodiments, Cas proteins can form components of inducible systems. The inducibility of the system will allow the use of some form of energy to control gene editing or gene expression in time and space. The form of the energy may include, but is not limited to, electromagnetic radiation, acoustic energy, chemical energy, and thermal energy. Examples of inducible systems include tetracycline-inducible promoters (Tet-On or Tet-Off), small molecule double hybrid transcription activation systems (FKBP, ABA, etc.), or light-inducible systems (plant phytochromes, LOV domains, or cryptochromes). In one embodiment, the CRISPR effector protein may be part of a light-inducible transcription effector (LITE) that directs changes in transcriptional activity in a sequence-specific manner. The components of light-inducible transcription effectors may include CRISPR effector proteins, light-responsive cytochrome heterodimers (e.g., from Arabidopsis thaliana), and transcriptional activation/repression domains. Further examples of inducible DNA binding proteins and methods of use thereof are provided in US 61/736465 and US 61/721,283 and WO 2014018423A2, which are incorporated herein by reference in their entireties.

In certain embodiments, the mutant Cas may have one or more mutations that result in reduced off-target effects, for example, the improved CRISPR enzyme modifies the target locus but reduces or eliminates off-target activity when complexed with the guide RNA, and the improved CRISPR enzyme increases CRISPR enzyme activity when complexed with the guide RNA. It should be understood that the mutant enzymes described below can be used in any of the methods described elsewhere herein according to the present disclosure. Any methods, products, compositions and uses described elsewhere herein are also applicable to the mutant CRISPR enzymes described in further detail below.

Methods and mutations that can be used in various combinations to increase or decrease the targeted activity and/or specificity relative to off-target activity, or to increase or decrease the targeted binding and/or specificity relative to off-target binding, can be used to compensate for or enhance mutations or modifications performed to promote other effects. Such mutations or modifications performed to promote other effects include mutations or modifications to Cas and/or mutations or modifications to guide RNA. The methods and mutations disclosed herein are used to regulate Cas nuclease activity and/or bind to chemically modified guide RNA.

In certain embodiments, the catalytic activity of the Cas protein of the present disclosure is changed or modified. It should be understood that if the catalytic activity is different from the catalytic activity of the corresponding wild-type Cas protein (e.g., an unmutated Cas protein), the mutated Cas has a changed or modified catalytic activity. The catalytic activity can be determined by means known in the art. For example, but not limited to, the catalytic activity can be determined in vitro or in vivo by measuring the insertion and deletion percentage (e.g., after a given time or at a given dose). In certain embodiments, the catalytic activity is improved. In certain embodiments, the catalytic activity is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100%. In certain embodiments, the catalytic activity is reduced. In certain embodiments, the catalytic activity is reduced by at least 5%, preferably at least 10%, more preferably at least 20%, for example, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or (substantially) 100%. One or more mutations herein can inactivate catalytic activity, which can significantly reduce all catalytic activity, reduce the activity to below detectable levels, or reduce it to no measurable catalytic activity.

One or more features of the engineered Cas protein may be different from the corresponding wild-type Cas protein. Examples of such features include catalytic activity, gRNA binding, specificity of Cas protein (e.g., editing the specificity of the defined target), stability of Cas protein, off-target binding, target binding, protease activity, nickase activity, PFS recognition. In some examples, the engineered Cas protein may include one or more mutations of the corresponding wild-type Cas protein. In some embodiments, the catalytic activity of the engineered Cas protein is improved compared with the corresponding wild-type Cas protein. In some embodiments, the catalytic activity of the engineered Cas protein is reduced compared with the corresponding wild-type Cas protein. In some embodiments, the gRNA binding of the engineered Cas protein is improved compared with the corresponding wild-type Cas protein. In some embodiments, the gRNA binding of the engineered Cas protein is reduced compared with the corresponding wild-type Cas protein. In some embodiments, the specificity of the Cas protein is improved compared with the corresponding wild-type Cas protein. In some embodiments, the specificity of the Cas protein is reduced compared with the corresponding wild-type Cas protein. In some embodiments, the stability of the Cas protein is improved compared with the corresponding wild-type Cas protein. In some embodiments, the stability of the Cas protein is reduced compared to the corresponding wild-type Cas protein. In some embodiments, the engineered Cas protein further comprises one or more mutations that inactivate catalytic activity. In some embodiments, the off-target binding of the Cas protein is improved compared to the corresponding wild-type Cas protein. In some embodiments, the off-target binding of the Cas protein is reduced compared to the corresponding wild-type Cas protein. In some embodiments, the target binding of the Cas protein is improved compared to the corresponding wild-type Cas protein. In some embodiments, the target binding of the Cas protein is reduced compared to the corresponding wild-type Cas protein. In some embodiments, the engineered Cas protein has higher protease activity or polynucleotide binding ability compared to the corresponding wild-type Cas protein. In some embodiments, PFS recognition is changed compared to the corresponding wild-type Cas protein.

Examples of Cas proteins

Examples of Cas proteins include Class I (e.g., Type I, Type III, and Type IV) and Class II (e.g., Type II, Type V, and Type VI) Cas proteins, such as Cas9, Cas12 (e.g., Cas12a, Cas12b, Cas12c, Cas12d), Cas13 (e.g., Cas13a, Cas13b, Cas13c, Cas13d), CasX, CasY, Cas14, variants thereof (e.g., mutant forms, truncated forms), homologs thereof, and orthologs thereof. The terms "orthologs" and "homologs" are well known in the art. By further guidance, a "homolog" of a protein used herein is a protein of the same species that performs the same or similar function as a protein as its homolog. Homologous proteins may, but are not necessarily, structurally related, or only partially related in structure. A "ortholog" of a protein used herein is a protein of a different species that performs the same or similar function as a protein as its ortholog. Orthologous proteins may, but are not necessarily, structurally related, or may be only partially related in structure.

Class 2 Cas proteins

In some embodiments, the Cas protein is a Class 2 Cas protein, i.e., a Class 2 CRISPR-Cas system Cas protein. Class 2 CRISPR-Cas systems can be subtypes, such as Type II-A, Type II-B, Type II-C, Type V-A, Type V-B, Type V-C, or Type V-U. In some embodiments, the Cas protein is Cas9, Cas12a, Cas12b, Cas12c, or Cas12d. In some embodiments, Cas9 can be SpCas9, SaCas9, StCas9, and other Cas9 orthologs. Cas12 can be Cas12a, Cas12b, and Cas12c, including FnCas12a, or homologs or orthologs thereof. Definitions and exemplary members of CRISPR-Cas systems include those described in Kira S. Makarova and Eugene V. Koonin, Annotation and Classification of CRISPR-Cas systems, Methods Mol Biol. 2015; 1311: 47-75; and Sergey Shmakov et al., Diversity and evolution of class 2 CRISPR-Cas systems, Nat Rev Microbial. 2017 Mar; 15(3): 169-182.

Cas protein adaptors

In some instances, the Cas protein includes at least one RuvC domain and at least one HNH domain. The Cas protein may further include the first and second joint domains connecting the RuvC domain and the HNH domain. The first joint (L1) and the second joint (L2) connecting the HNH and RuvC domains in Cas9 are described in the following studies: Nishimasu, H. et al. "Crystal structure of Cas9 in complex with guide RNA and target RNA" Cell 156 (Feb. 27, 2014): 935-949 and Ribeiro, L. et al. (2018) "Protein engineering strategies to expand CRISPR-Cas9 applications" International Journal of Genomics Volume 2018, Article ID 1652567 (doi.org/10.1155/2018/1652567). Ribeiro's Figure 1 shows the overall organization, structure and function of Cas9, which is specifically incorporated herein by reference. Specifically, Figure 1A shows a schematic diagram of the domain organization of SpCas9, indicating the genetic structure of the HNH and RuvC domains including the linkers L1 (spanning amino acids 765-780) and L2 (spanning amino acids 906-918) described herein.

Similarly, when referencing the first and second linker domains, the domain organization of Staphylococcus aureus Cas9 (SaCas9) can be utilized. In one aspect, the linker 1 domain region spans residues 481-519 and connects the RuvC-II domain to the HNH domain in SaCas9. In certain embodiments, the linker 2 region spans residues 629-649 and connects the RuvC-III domain and the HNH domain of SaCas9. Therefore, the first and/or second linker domains in the Cas9 orthologs can be mutated, and reference can be made to the amino acid residues corresponding to the amino acids of wild-type SaCas9. See, Nishimasu, Cell. 2015 Aug 27; 162 (5): 1113-1126; doi: 10.1016 / j.cell.2015.08.007, which is incorporated herein by reference. Specifically, Figures 1, S1-S3 of Nishimasu describe the domain organization of the Cas9 protein in detail and are specifically incorporated herein by reference.

The first and second joints can comprise about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or more amino acids. The first and second joints can correspond to wild-type joints. In some aspects, the first and second joints can comprise one or more mutations in the first and/or second joints. In one aspect, the first and/or second joints comprise one or more mutations that improve the specificity of the Cas9 protein.

In certain embodiments, the linkers L1 and L2 connecting the HNH and RuvC domains of Cas9 contain wild-type amino acid sequences. In certain embodiments, the linkers connecting the HNH and RuvC domains contain mutations in one or more amino acids. In an exemplary embodiment, the first linker (L1) contains a mutation corresponding to the amino acid T769I of SpCas9, and/or the second linker (L2) contains a mutation corresponding to the amino acid G915M of SpCas9. In an exemplary embodiment, one or more linker mutations such as T769I and G915M confer improved specificity to the Cas9 protein.

In one embodiment, one or more mutations in the first and second linkers can be combined with one or more mutations in other parts of the Cas9 protein to further improve specificity and/or maintain activity substantially equivalent to that of a wild-type Cas9 protein, as described herein. In one embodiment, mutations in the linkers and/or additional mutations within the Cas protein can be identified using the methods detailed herein that enhance/improve specificity and substantially maintain the wild-type activity of a wild-type Cas9.

2. Type II Cas proteins (e.g. Cas9)

In some embodiments, the Cas protein can be a Cas protein (Type II Cas protein) of a Type II CRISPR-Cas system of type 2. In some embodiments, the Cas protein can be a Type II Cas protein of type 2, such as Cas9. In some embodiments, the system based on CRISPR/Cas9 can include a Cas9 protein or a fragment thereof, a Cas9 fusion protein, a nucleic acid encoding a Cas9 protein or a fragment thereof, or a nucleic acid encoding a Cas9 fusion protein. The so-called "Cas9 (CRISPR-associated protein 9)" refers to a polypeptide or a fragment thereof having at least about 85% amino acid identity with NCBI accession number NP_269215 and having RNA binding activity, DNA binding activity and/or DNA cleavage activity (e.g., endonuclease or nickase activity). "Cas9 function" can be defined by any one of a variety of assays, including but not limited to a nucleic acid binding assay based on fluorescence polarization, a fluorescence polarization chain invasion assay, a transcription assay, an EGFP destruction assay, a DNA cleavage assay, and/or a Surveyor assay, such as described herein. The so-called "Cas 9 nucleic acid molecule" refers to a polynucleotide encoding a Cas 9 polypeptide or a fragment thereof. Exemplary Cas9 nucleic acid molecule sequences are provided at genomic sequence number NC_002737. In certain embodiments, disclosed herein are inhibitors of Cas9 or its variants naturally occurring in Cas9, such as Streptococcus pyogenes (S.pyogenes) (SpCas9) or Staphylococcus aureus (S.aureus) (SaCas9). Cas9 uses the base pairing of the protoplast adjacent motif (PAM) sequence and guide RNA (gRNA) with the target DNA to identify foreign DNA. The relative ease with which Cas9 induces targeted chain breaks at any genomic site enables efficient genome editing in a variety of cell types and organisms. Cas9 derivatives can also be used as transcriptional activators/repressors.

In some cases, the CRISPR-Cas protein is Cas9 or a variant thereof. In some instances, Cas9 can be wild-type Cas9, including any naturally occurring bacterial Cas9. Cas9 orthologs generally have a general organization of 3-4 RuvC domains and one HNH domain. The RuvC domain closest to the 5' end cuts the non-complementary chain, and the HNH domain cuts the complementary chain. All symbols refer to the guide sequence. The catalytic residues in the 5' RuvC domain are identified by comparing the Cas9 of interest with other Cas9 orthologs (from S.pyogenes type II CRISPR loci, S.thermophilus CRISPR loci 1, S.thermophilus CRISPR loci 3, and Franciscilla novicida type II CRISPR loci), and the conserved Asp residue (D10) is mutated to alanine to convert Cas9 into a complementary chain cutting enzyme. Therefore, the Cas enzyme can be wild-type Cas9, including any naturally occurring bacterial Cas9. CRISPR, Cas or Cas9 enzymes can be codon-optimized, or modified versions, including any chimeras, mutants, homologs or orthologs. In another aspect of the present disclosure, the Cas9 enzyme can contain one or more mutations and can be used as a universal DNA binding protein with or without fusion to a functional domain.

The mutation may be an artificially introduced mutation or a gain-of-function or loss-of-function mutation. In certain embodiments, the transcriptional activation domain may be VP64. In certain embodiments, the transcriptional repressor domain may be KRAB or SID4X. Other aspects of the disclosure relate to mutated Cas9 enzymes fused to domains, including but not limited to nucleases, transcriptional activators, repressors, recombinases, transposases, histone remodelers, demethylases, DNA methyltransferases, cryptochromes, light-induced/controllable domains, or chemically induced/controllable domains. The disclosure may relate to guides or chimeric guide sequences that allow sgRNA or tracrRNA or allow enhancement of the performance of these RNAs in cells. This type II CRISPR enzyme may be any Cas enzyme. In some cases, the Cas9 enzyme is from or derived from SpCas9 or SaCas9. The term "derived" as used herein refers to an enzyme that is largely based on a wild-type enzyme (in the sense of having a high degree of sequence homology to the wild-type enzyme), but has been mutated (modified) in a manner known in the art or described herein. In one example, the mutation may include one or more mutations in the first connecting domain, the second connecting domain, and/or other portions of the protein. A high degree of sequence homology may include at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more relative to the wild-type enzyme.

The Cas enzyme may be an identified Cas9, as this may refer to a general category of enzymes that share homology with the largest nuclease with multiple nuclease domains from a type II CRISPR system. In some cases, the Cas9 enzyme is from or derived from SpCas9 (Streptococcus pyogenes (S.pyogenes) Cas9) or saCas9 (Staphylococcus aureus (S.aureus) Cas9). "StCas9" refers to wild-type Cas9 (UniProt ID: G3ECR1) from Streptococcus thermophilus (S.thermophilus). Similarly, "SpCas9" refers to wild-type Cas9 (UniProtID: Q99ZW2) from Streptococcus pyogenes (S.pyogenes). The term "derived" as used herein refers to an enzyme that is largely based on a wild-type enzyme (in the sense of having a high degree of sequence homology with the wild-type enzyme), but it has been mutated (modified) in a manner known in the art or described herein. It should be understood that the terms Cas and CRISPR enzymes are generally used interchangeably herein unless otherwise expressly stated. As noted above, many of the residue numbers used herein refer to the Cas9 enzyme from a type II CRISPR locus in Streptococcus pyogenes.

In a specific embodiment, the effector protein is a Cas9 effector protein from or derived from an organism of the genera Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibac ter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacill us), Brevibacilus, Methylobacterium or Acidaminococcus, Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Sutterella, Legionella ella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma, or Campylobacter.

In certain embodiments, the Cas9 protein is from or derived from an organism selected from the group consisting of: Streptococcus mutans, Streptococcus agalactiae, Streptococcus equisimilis, Streptococcus sanguinis, Streptococcus pneumoniae, Campylobacter jejuni, Campylobacter coli, N. salsuginis, N. tergarcus, Staphylococcus auricularis, Staphylococcus carnosus, Neisseria meningitidis, Neisseria gonorrhoeae. gonorrhoeae), Listeria monocytogenes, Listeria ivanovii, C. botulinum, C. difficile, C. tetani or C. sordellii, Francisella tularensis 1, Francisella tularensis subsp. novicida, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011 GWA2_33_10, Parcubacteria bacterium GW2011 GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira in adai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens and Porphyromonas macacae. In certain embodiments, the Cas9 protein is a Cas9 from or derived from the organism Streptococcus pyogenes, Staphylococcus aureus, or Streptococcus thermophilus.

In a more preferred embodiment, the Cas9 protein is derived from a bacterial species selected from Streptococcus pyogenes, Staphylococcus aureus or Streptococcus thermophilus. In certain embodiments, the Cas9 is derived from a bacterial species selected from the group consisting of Francisella tularensis 1, Prevotella albensis, Lachnospiraceae bacterium MC20171, Butyrivibrioproteoclasticus, Peregrinibacteria bacterium GW2011 GWA2 33JO, Parcubacteria bacterium GW2011 GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasmatermitum), Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotelladisiens and Porphyromonas macacae. In certain embodiments, the Cas9 protein is derived from a bacterial species selected from Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium MA2020. In certain embodiments, the effector protein is derived from a subspecies of Francisella tularensis 1, including but not limited to Francisella tularensis subsp. novicida.

Cas9 enzymes include, but are not limited to, Streptococcus pyogenes serotype M1 (UniProt ID: Q99ZW2), Staphylococcus aureus Cas9 (UniProt ID: J7RUA5), Eubacterium ventriosum Cas9 (UniProt ID: A5Z395), Azospirillum (strain B510) Cas9 (UniProt ID: D3NT09), Gluconacetobacter diazotrophicus (strain ATCC 49037) Cas9 (UnitProt ID: A9HKP2), Neisseria cinerea Cas9 (UniProt ID: D0W2Z9), Roseburia intestinalis Cas9 (UniProt ID: C7G697), Parvibaculum lavamentivorans) (strain DS-1) Cas9 (UniProt ID: A7HP89), Nitratifractor salsuginis (strain DSM 16511) Cas9 (UniProt ID: E6WZS9), Campylobacter lari Cas9 (UniProt ID: G1UFN3).

The enzyme action of Cas9 derived from Streptococcus pyogenes or any closely related Cas9 produces a double-strand break at the target site sequence, which is hybridized to 20 nucleotides of the guide sequence and has a pre-spacer adjacent motif (PAM) sequence (examples include NGG/NRG or a PAM that can be determined as described herein) after the 20 nucleotides of the target sequence. The CRISPR activity of site-specific DNA recognition and cleavage by Cas9 is defined by the guide sequence, the tracr sequence that partially hybridizes to the guide sequence, and the PAM sequence. More aspects of the CRISPR system are described in Karginov and Hannon, The CRISPR system: small RNA-guided defense in bacteria and archaea, Mole Cell 2010, January 15; 37(1):7. The type II CRISPR locus from Streptococcus pyogenes SF370 contains a cluster of four genes Cas9, Cas1, Cas2 and Csnl, as well as two non-coding RNA elements tracrRNA and a characteristic array of repetitive sequences (forward repeats) separated by short non-repetitive sequences (spacers, each about 30bp). In this system, targeted DNA double-strand breaks (DSBs) are produced in four consecutive steps. First, two non-coding RNAs, i.e., pre-crRNA arrays and tracrRNA, are transcribed from the CRISPR locus. Secondly, tracrRNA hybridizes with the forward repeats of pre-crRNA and is then processed into mature crRNA containing each spacer sequence. Third, mature crRNA:tracrRNA complexes are formed by heteroduplexes between the crRNA spacer and the pre-spacer DNA, and Cas9 is guided to a DNA target consisting of a pre-spacer sequence and a corresponding PAM. Finally, Cas9 mediates the cutting of the target DNA upstream of the PAM, producing DSBs in the pre-spacer sequence. The pre-crRNA array consisting of a single spacer with two forward repeats (DRs) on both sides is also covered by the term "tracr-mate sequence". In certain embodiments, Cas9 can be constitutively present or inducibly present or conditionally present or administered or delivered. Cas9 optimization can be used to enhance function or develop new functions. Chimeric Cas9 proteins can be produced, and Cas9 can be used as a universal DNA binding protein. The structural information provided for Cas9 can be used to further engineer and optimize the CRISPR-Cas system, and this can also infer the structure-function relationship of other CRISPR enzyme systems, particularly other type II CRISPR enzymes or Cas9 orthologs. Structure-function relationships. Crystal structure information (described in U.S. Provisional Applications 61/915,251, filed December 12, 2013; 61/930,214, filed January 22, 2014; 61/980,012, filed April 15, 2014; and Nishimasu et al, "Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA," Cell 156(5):935-949, DOI: http://dx.doi.org/10.1016/j.cell.2014.02.001 (2014), each of which is incorporated herein by reference in its entirety) provides structural information for truncating and generating modular or multipartite CRISPR enzymes that can be incorporated into inducible CRISPR-Cas systems. Specifically, structural information for Streptococcus pyogenes (S. pyogenes) Cas9 (SpCas9) is provided, and this can be extrapolated to other Cas9 orthologs or other type II CRISPR enzymes. The Cas9 gene is present in several different bacterial genomes, often in the same locus as the cas1, cas2, and cas4 genes and the CRISPR box. In addition, the Cas9 protein contains an easily identifiable C-terminal region that is homologous to the transposon ORF-B and includes an active RuvC-like nuclease, an arginine-rich region.

dCas9

The Cas9 protein can be mutated to inactivate nuclease activity. An inactivated Cas9 protein (iCas9, also referred to as "dCas9") from Streptococcus pyogenes (S.pyogenes) without endonuclease activity has recently been targeted by gRNA to genes in bacteria, yeast and human cells to silence gene expression by steric hindrance. "DCas molecule" used herein may refer to dCas protein or a fragment thereof. "DCas9 molecule" used herein may refer to dCas9 protein or a fragment thereof. The terms "iCas" and "dCas" used herein are used interchangeably and refer to catalytically inactivated CRISPR-associated proteins. In one embodiment, the dCas molecule includes one or more mutations in the DNA cleavage domain. In one embodiment, the dCas molecule includes one or more mutations in the RuvC or HNH domain. In one embodiment, the dCas molecule includes one or more mutations in both the RuvC and HNH domains. In one embodiment, the dCas molecule is a fragment of a wild-type Cas molecule. In one embodiment, the dCas molecule comprises a functional domain from a wild-type Cas molecule, wherein the functional domain is selected from a Reel domain, a bridge helical domain, or a PAM interaction domain. In one embodiment, the nuclease activity of the dCas molecule is reduced by at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% compared to the corresponding wild-type Cas molecule.

Suitable dCas molecules can be derived from wild-type Cas molecules. The Cas molecules can be from type I, type II or type III CRISPR-Cas systems. In one embodiment, suitable dCas molecules can be derived from Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 or Cas10 molecules. In one embodiment, the dCas molecules are derived from Cas9 molecules. The dCas9 molecules can be obtained by, for example, introducing point mutations (e.g., substitutions, deletions or additions) in DNA cleavage domains such as nuclease domains, such as RuvC and/or HNH domains in Cas9 molecules. See, for example, Jinek et al., Science (2012) 337: 816-21, which is incorporated herein by reference in its entirety. For example, introducing two point mutations in RuvC and HNH domains reduces Cas9 nuclease activity while retaining Cas9 sgRNA and DNA binding activity. In one embodiment, the two point mutations in the RuvC and HNH active sites are D10A and H840A mutations of the S. pyogenes Cas9 molecule. Alternatively, D10 and H840 of the Cas9 molecule can be deleted to inactivate the Cas9 nuclease activity while retaining its sgRNA and DNA binding activity. In one embodiment, the two point mutations in the RuvC and HNH active sites are D10A and N580A mutations of the S. pyogenes Cas9 molecule.

In one embodiment, the dCas molecule is a Staphylococcus aureus (S. aureus) dCas9 molecule numbered according to SEQ ID NO: 1, comprising mutations at D10 and/or N580. In one embodiment, the dCas molecule is a Staphylococcus aureus (S. aureus) dCas9 molecule numbered according to SEQ ID NO: 1, comprising D10A and/or N580A mutations.

Staphylococcus aureus (S. aureus) dCas9

In one embodiment, the dCas9 molecule is a Staphylococcus aureus (S. aureus) dCas9 molecule, which comprises the amino acid sequence of SEQ ID NO: 2 or 3, a sequence substantially identical to SEQ ID NO: 2 or 3 (e.g., a sequence identity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more), or a sequence having 1, 2, 3, 4, 5 or more changes (e.g., amino acid substitutions, insertions or deletions) relative to SEQ ID NO: 2 or 3, or any fragment thereof.

Similar mutations can also be applied to any other naturally occurring Cas9 (e.g., Cas9 from other species) or engineered Cas9 molecules. In certain embodiments, the dCas9 molecule includes a Streptococcus pyogenes dCas9 molecule, a Staphylococcus aureus dCas9 molecule, a Campylobacter jejuni dCas9 molecule, a Corynebacterium diphtheria dCas9 molecule, an Eubacterium ventriosum dCas9 molecule, a Streptococcus pasteurianus dCas9 molecule, a Lactobacillus farciminis dCas9 molecule, a Sphaerochaeta globus dCas9 molecule, an Azospirillum (strain B510) dCas9 molecule, a Gluconacetobacter diazotrophicus dCas9 molecule, a Neisseria griseus dCas9 molecule, a cinerea) dCas9 molecule, Roseburia intestinalis dCas9 molecule, Parvibaculum lavamentivorans dCas9 molecule, Nitratifractor salsuginis (strain DSM 16511) dCas9 molecule, Campylobacterlari (strain CF89-12) dCas9 molecule, Streptococcus thermophilus (strain LMD-9) dCas9 molecule or its fragments.

In certain embodiments, the present disclosure provides a vector comprising a dCas9 molecule encoding Streptococcus pyogenes, Staphylococcus aureus, Campylobacter jejuni, Corynebacterium diphtheria, Eubacterium ventriosum, Streptococcus pasteurianus, Lactobacillus farciminis, Sphaerochaeta globus, Azospirillum (strain B510), Gluconacetobacter diazotrophicus, Neisseria griseus, and the like. cinerea) dCas9 molecule, Roseburia intestinalis dCas9 molecule, Parvibaculum lavamentivorans dCas9 molecule, Nitratifractor salsuginis (strain DSM 16511) dCas9 molecule, Campylobacter lari (strain CF89-12) dCas9 molecule, Streptococcus thermophilus (strain LMD-9) dCas9 molecule or nucleotides of their fragments.

Exemplary dCas9 proteins include, but are not limited to, those listed in Table 1.

Table 1. Exemplary dCas9 proteins

Cas9 fusion protein

The CRISPR/Cas9-based system may include a fusion molecule (e.g., DNMT3A-DNMT3L (3A3L)-dCas9-KRAB). The fusion molecule may include at least one DNA binding protein (e.g., dCas9) and at least one gene expression regulator (e.g., KRAB, DNMT3A, DNMT3L, DNMT3A-DNMT3L fusion peptide). In certain embodiments, the gene expression regulator is selected from a repressor of gene expression (e.g., KRAB), a gene expression activator, or an epigenetic modification regulator (e.g., DNMT3A, DNMT3L, DNMT3A-DNMT3L fusion peptide) or a combination thereof. Different regulators of gene expression are known in the art, see, e.g., Thakore et al., Nat Methods. 2016; 13: 127-37, which is incorporated herein by reference in its entirety.

Repressors of gene expression

In certain embodiments, the gene expression regulator comprises a repressor of gene expression. The repressor may be any known gene expression repressor, for example, selected from Kruppel-associated box (KRAB) domain, mSin3 interaction domain (SID), MAX interacting protein 1 (MXI1), chromosome shadow domain, EAR repression domain (SRDX), eukaryotic release factor 1 (ERF1), eukaryotic release factor 3 (ERF3), tetracycline repressor, lad repressor, Catharanthus roseus G-box binding factor 1 and 2, Drosophila Groucho, Tripartite motif-containing 28 (TRTM28), nuclear receptor corepressor 1, nuclear receptor corepressor 2 repressor or a fragment or fusion thereof.

Kruppel Related Box (KRAB)

The KRAB domain is a transcriptional repressor domain present in the N-terminal portion of many zinc finger protein-based transcription factors. The KRAB domain functions as a transcriptional repressor when it binds to target DNA through the DNA binding domain. The KRAB domain is rich in charged amino acids and can be divided into subdomains A and B. The KRAB A and B subdomains can be separated by a variable spacer, and many KRAB proteins contain only the A subdomain. A 45-amino acid sequence in the KRAB A subdomain has been shown to be important for transcriptional repression. The B subdomain does not repress transcription itself, but enhances the repressive effect of the KRAB A subdomain. The KRAB domain recruits the corepressors KAP1 (KRAB-associated protein-1, also known as transcription mediator 1β, KRAB-A interacting protein, and ternary motif protein 28) and heterochromatin protein 1 (HP1), as well as other chromatin regulatory proteins, causing transcriptional repression through the formation of heterochromatin. In one embodiment, the methods and compositions disclosed herein include a fusion molecule comprising a dCas9 molecule fused to a KRAB domain or a fragment thereof. In one embodiment, the KRAB domain or a fragment thereof is fused to the N-terminus of the dCas9 molecule. In one embodiment, the KRAB domain or a fragment thereof is fused to the C-terminus of the dCas9 molecule. In one embodiment, the KRAB domain or a fragment thereof is fused to both the N-terminus and the C-terminus of the dCas9 molecule. In one embodiment, the KRAB domain or a fragment thereof is fused to both the N-terminus and the C-terminus of the dCas9 molecule. In one embodiment, the fusion molecule comprises a KRAB domain comprising a sequence of SEQ ID NO: 22, a sequence substantially identical to SEQ ID NO: 22 (e.g., having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity), or a sequence having 1, 2, 3, 4, 5 or more changes (e.g., amino acid substitutions, insertions or deletions) relative to SEQ ID NO: 22, or any fragment thereof.

Exemplary KRAB domain sequences

mSin3 interaction domain (SID)

The mSin3 interaction domain (SID) is an interaction domain present on several transcriptional repressor proteins. It interacts with the paired amphipathic α-helix 2 (PAH2) domain of mSin3, which is a transcriptional repressor domain attached to a transcriptional repressor protein such as an mSin3 A co-repressor. In one embodiment, the methods and compositions disclosed herein include a fusion molecule comprising a dCas9 molecule fused to an mSin3 interaction domain or a fragment thereof. In one embodiment, the methods and compositions disclosed herein include a fusion molecule comprising a dCas9 molecule fused to four tandem mSin3 interaction domains (SID4X). In one embodiment, the four tandem mSin3 interaction domains (SID4X) are fused to the C-terminus of the dCas9 molecule.

MAX interacting protein 1 (MXI1)

Mxi1 is a repressor of MYC. Mxi1 may antagonize MYC transcriptional activity by competing for binding to MYC-associated factor X (MAX), which binds to MYC and is necessary for MYC to function. In one embodiment, the methods and compositions disclosed herein include a fusion molecule comprising a dCas9 molecule fused to Mxi1 or a fragment thereof. In one embodiment, Mxi1 is fused to the C-terminus of the dCas9 molecule.

Gene expression activator

In some embodiments, the gene expression regulator includes a gene expression activator. The activator can be any known gene expression activator, such as a VP16 activation domain, a VP64 activation domain, a p65 activation domain, an Epstein-Barr virus (Epstein-Barr virus) R transactivator Rta molecule or a fragment thereof. Activators that can be used with dCas9 molecules are known in the art. See, for example, Chavez et al., Nat Methods. (2016) 13: 563-67, which is incorporated herein by reference in its entirety.

VP16, VP64, VP160

VP16 is a 16-amino acid viral protein sequence that recruits transcriptional activators to promoters and enhancers. VP64 is a transcriptional activator comprising four copies of VP16, for example, a molecule comprising four tandem copies of VP16 connected by a Gly-Ser linker. VP160 is a transcriptional activator comprising 10 copies of VP16. In one embodiment, the methods and compositions disclosed herein include fusion molecules comprising dCas9 molecules fused to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of VP16. In one embodiment, the methods and compositions disclosed herein include fusion molecules comprising dCas9 molecules fused to VP64. In one embodiment, the methods and compositions disclosed herein include fusion molecules comprising dCas9 molecules fused to VP160. In one embodiment, VP64 is fused to the C-terminus, N-terminus, or both the N-terminus and the C-terminus of the dCas9 molecule.

p65 activation domain (p65AD)

p65AD is the major transactivation domain of the 65 kDa polypeptide of the nuclear form of the F-κΒ transcription factor. An exemplary sequence of the human transcription factor p65 can be obtained in the Uniprot database under accession number Q04206. In one embodiment, the methods and compositions disclosed herein include a fusion molecule comprising a dCas9 molecule fused to p65 or a fragment thereof, such as p65AD.

Epstein-Barr virus (EBV) R transactivator (Rta)

Rta, the immediate early protein of EBV, is a transcriptional activator that induces lytic gene expression and triggers viral reactivation. In one embodiment, the methods and compositions disclosed herein include a fusion molecule comprising a dCas9 molecule fused to Rta or a fragment thereof.

VP64, p65, Rta fusion

In one embodiment, the methods and compositions disclosed herein include a fusion molecule comprising a dCas9 molecule fused to VP64, p65, Rta, or any combination thereof. The ternary activator VP64-p65-Rta (also referred to as VPR), in which the three transcriptional activation domains are fused using a short amino acid linker, can effectively upregulate target gene expression when fused to a dCas9 molecule. In one embodiment, the methods and compositions disclosed herein include a dCas9 molecule fused to a VPR.

Synergistic activation mediator (SAM)

In one embodiment, the methods and compositions disclosed herein include a CRISPR-Cas system comprising three components: (1) a dCas9-VP64 fusion, (2) a gRNA incorporating two MS2 RNA aptamers at the tetraloop and stem loop, and (3) an MS2-P65-HSF1 activation accessory protein. This system, named Synergistic Activation Mediator (SAM), brings together three activation domains—VP64, P65, and HSF1, and has been described in Konermann et al., Nature. 2015; 517: 583-8, which is incorporated herein by reference in its entirety.

Ldbl self-association domain

In one embodiment, the methods and compositions disclosed herein include a fusion molecule comprising a dCas9 molecule fused to a Ldbl self-association domain. The Ldbl self-association domain recruits endogenous Ldbl associated with an enhancer.

Epigenetic Modifiers

In one embodiment, methods and compositions disclosed herein include fusion molecules, which include dCas9 molecules fused to gene expression regulators. In certain embodiments, the gene expression regulator includes an epigenetically modified regulator. In one embodiment, the fusion molecule is modified by epigenetics, such as by histone acetylation or methylation or DNA methylation at a regulatory element of a target gene such as a promoter, an enhancer, or a transcription start site, to regulate target gene expression. The regulator can be any known epigenetic modification regulator, such as histone acetyltransferase (such as p300 catalytic domain), histone deacetylase, histone methyltransferase (such as SUV39H1 or G9a (EHMT2)), histone demethylase (such as LSD1), DNA methyltransferase (such as DNMT3a or DNMT3a-DNMT3L), DNA demethylase (such as TET1 catalytic domain or TDG) or a fragment thereof.

Histone modification activity

In certain embodiments, the epigenetic modification regulator may have histone modification activity. Histone modification activity may include but is not limited to histone deacetylase, histone acetyltransferase, histone demethylase or histone methyltransferase activity.

In some embodiments, the epigenetic modification regulator may have histone acetyltransferase activity. The histone acetyltransferase may be p300 or CREB binding protein (CBP) protein or its fragment. In some embodiments, the methods and compositions disclosed herein include fusion molecules, which include dCas9 molecules fused to the catalytic core of acetyltransferase p300 or its fragment such as p300. In some embodiments, the methods and compositions disclosed herein include fusion molecules, which include dCas9 molecules fused to CREB binding protein (CBP) protein or its fragment.

In some embodiments, the epigenetic modification regulator may have histone demethylase activity. For example, the epigenetic modification regulator may include an enzyme that removes a methyl (CH3-) group from a nucleic acid or protein (e.g., histone). In some embodiments, the methods and compositions disclosed herein include a fusion molecule comprising a dCas9 molecule fused to a Lys-specific histone demethylase 1 (LSD1) or a fragment thereof.

In certain embodiments, the epigenetic modification regulator may have histone methyltransferase activity. In certain embodiments, the methods and compositions disclosed herein include a fusion molecule comprising a dCas9 molecule fused to SUV39H1 or a fragment thereof. In certain embodiments, the methods and compositions disclosed herein include a fusion molecule comprising a dCas9 molecule fused to G9a (EHMT2) or a fragment thereof.

DNA demethylase activity

In some embodiments, the epigenetic modification regulator may have DNA demethylase activity. For example, the epigenetic modification regulator may convert methyl to hydroxymethylcytosine as a mechanism for DNA demethylation. In some embodiments, the methods and compositions disclosed herein include a fusion molecule comprising a dCas9 molecule fused to 10-11 translocation methylcytosine dioxygenase 1 (TET1) or a fragment thereof. In some embodiments, the methods and compositions disclosed herein include a fusion molecule comprising a dCas9 molecule fused to thymine DNA glycosidase (TDG) or a fragment thereof.

DNA methylase activity

In certain embodiments, the epigenetic modification regulator may have DNA methylase activity. For example, the epigenetic modification regulator may have methylase activity, which involves transferring methyl to DNA, RNA, protein, small molecule, cytosine or adenine. In certain embodiments, the methods and compositions disclosed herein include fusion molecules comprising dCas9 molecules fused to DNMT3A or a fragment thereof. In certain embodiments, the methods and compositions disclosed herein include fusion molecules comprising dCas9 molecules fused to DNMT3L or a fragment thereof. In certain embodiments, the methods and compositions disclosed herein include fusion molecules comprising dCas9 molecules fused to DNMT3L and DNMT3L or a fragment thereof. In certain embodiments, the methods and compositions disclosed herein include fusion molecules comprising dCas9 molecules fused to DNMT3A-DNMT3L fusion peptides.

DNMT3A

DNMT3L

DNMT3A-DNMT3L fusion peptide

In one embodiment, the Cas9 fusion protein further comprises a nuclear localization sequence (NLS), such as a LS fused to the N-terminus and/or C-terminus of Cas9.

Nuclear localization sequences are known in the art. In one embodiment, the NLS comprises an amino acid sequence of SEQ ID NO: 25 or 26, a sequence substantially identical to SEQ ID NO: 25 or 26 (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical), or a sequence having 1, 2, 3, 4, 5 or more changes (e.g., amino acid substitutions, insertions or deletions) relative to SEQ ID NO: 25 or 26, or any fragment thereof.

SEQ ID NO: 25 (Exemplary nuclear localization sequence)

APKKKRKVGIHGVPAA

SEQ ID NO: 26 (Exemplary nuclear localization sequence)

KRPAATKKAGQAKKKK

In some embodiments, the system based on CRISPR/Cas9 may include a dCas9 molecule and a gene expression regulator or a nucleic acid encoding a dCas9 molecule and a gene expression regulator. In one embodiment, the dCas9 molecule and the gene expression regulator are covalently linked. In one embodiment, the gene expression regulator is directly covalently fused to the dCas9 molecule. In one embodiment, the gene expression regulator is indirectly covalently fused to the dCas9 molecule, for example, by a non-regulator or a joint or by a second regulator. In one embodiment, the gene expression regulator is located at the N-terminus and/or C-terminus of the dCas9 molecule. In one embodiment, the dCas9 molecule and the gene expression regulator are non-covalently linked. Exemplary sequences include but are not limited to those listed in Table 2. In some embodiments, the joint between the dCas9 and at least one gene expression regulator comprises an amino acid sequence corresponding to the joint listed in Table 2.

Table 2. Exemplary linker sequences

In one embodiment, the dCas9 molecule is fused to a first tag, such as a first peptide tag. In one embodiment, the gene expression regulator is fused to a second tag, such as a second peptide tag. In one embodiment, the first and second tags, such as a first peptide tag and a second peptide tag, interact with each other non-covalently, thereby bringing the dCas9 molecule and the gene expression regulator into close proximity.

In one embodiment, the CRISPR/Cas9-based system comprises a fusion molecule or a nucleic acid encoding a fusion molecule. In one embodiment, the fusion molecule comprises a sequence comprising dCas9 fused to a gene expression regulator. In one embodiment, the dCas9 molecule includes a Streptococcus pyogenes dCas9 molecule, a Staphylococcus aureus dCas9 molecule, a Campylobacter jejuni dCas9 molecule, a Corynebacterium diphtheria dCas9 molecule, an Eubacterium ventriosum dCas9 molecule, a Streptococcus pasteurianus dCas9 molecule, a Lactobacillus farciminis dCas9 molecule, a Sphaerochaeta globus dCas9 molecule, an Azospirillum (strain B510) dCas9 molecule, a Gluconacetobacter diazotrophicus dCas9 molecule, a Neisseria griseus dCas9 molecule, and a Streptococcus pasteurianus dCas9 molecule. cinerea) dCas9 molecule, Roseburia intestinalis dCas9 molecule, Parvibaculum lavamentivorans dCas9 molecule, Nitratifractor salsuginis (strain DSM16511) dCas9 molecule, Campylobacter lari (strain CF89-12) dCas9 molecule, Streptococcus thermophilus (strain LMD-9) dCas9 molecule or its fragment.

In one embodiment, the fusion molecule is a DNMT3A-DNMT3L (3A3L)-dCas9-KRAB fusion molecule, which comprises from N-terminus to C-terminus: a DNMT3A-DNMT3L fusion peptide (3A3L), a dCas9 peptide, and a KRAB peptide domain fused directly or indirectly (e.g., via a linker).

In one embodiment, the fusion molecule comprises a fusion molecule comprising the amino acid sequence of SEQ ID NO:97, a sequence substantially identical to SEQ ID NO:97 (e.g., a sequence identity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more), or a sequence having 1, 2, 3, 4, 5 or more changes (e.g., substitutions, insertions or deletions) relative to SEQ ID NO:97, or any fragment thereof.

DNMT3A-DNMT3L(3A3L)-dCas9-KRAB

gRNA

In the case of the CRISPR-Cas system, the term "guide sequence" as used herein includes any polynucleotide sequence that has sufficient complementarity with the target nucleic acid sequence to hybridize with the target nucleic acid sequence and guide the sequence-specific binding of the nucleic acid targeting complex to the target nucleic acid sequence. The guide sequence can form a duplex with the target sequence. The duplex can be a DNA duplex, an RNA duplex or an RNA/DNA duplex. The terms "guide molecule", "guide RNA" and "single guide RNA" are used interchangeably herein and refer to RNA-based molecules that can form a complex with the CRISPR-Cas protein and include a guide sequence that has sufficient complementarity with the target nucleic acid sequence to hybridize with the target nucleic acid sequence and guide the sequence-specific binding of the complex to the target nucleic acid sequence. As described herein, guide molecules or guide RNAs particularly encompass RNA-based molecules with one or more chemical modifications (e.g., by chemically linking two ribonucleotides or by replacing one or more ribonucleotides with one or more deoxyribonucleotides).

The guide molecule or guide RNA of the CRISPR-Cas protein can include a tracr-mate sequence (covering a "direct repeat sequence" in the case of an endogenous CRISPR system) and a guide sequence (also referred to as a "spacer" in the case of an endogenous CRISPR system). In certain embodiments, the CRISPR-Cas system or complex described herein does not include a tracr sequence and/or does not rely on the presence of a tracr sequence. In certain embodiments, the guide molecule may include, consist essentially of, or consist of a direct repeat sequence fused or linked to a guide sequence or a spacer sequence.

In general, CRISPR-Cas systems are characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence. In the context of CRISPR complex formation, a "target sequence" refers to a sequence to which a guide sequence is designed to have complementarity, wherein hybridization between a target DNA sequence and a guide sequence promotes the formation of a CRISPR complex.

In some embodiments, the guide sequence or spacer length of the guide molecule is 15 to 50 nucleotides. In some embodiments, the spacer length of the guide RNA is at least 15 nucleotides in length. In some embodiments, the spacer length is 15 to 17 nucleotides, 17 to 20 nucleotides, 20 to 24 nucleotides, 23 to 25 nucleotides, 24 to 27 nucleotides, 27 to 30 nucleotides, 30 to 35 nucleotides, or greater than 35 nucleotides.

In some embodiments, the length of the guide sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 , 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 nucleotides.

In some embodiments, the sequence of the guide molecule (direct repeat sequence and/or spacer) is selected to reduce the degree of secondary structure in the guide molecule. In some embodiments, when optimally folded, the guide RNA of the target nucleic acid is equal to or less than about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1% or less nucleotides participate in self-complementary base pairing. Optimal folding can be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimum Gibbs free energy. An example of such an algorithm is mFold, as described in Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another exemplary folding algorithm is the online web server RNAfold, which was developed at the Institute of Theoretical Chemistry at the University of Vienna using the centroid structure prediction algorithm (see, e.g., A. R. Gruber et al., 2008, Cell 106(1):23-24 and PA Carr and GM Church, 2009, Nature Biotechnology 27(12):1151-62).

As described above, the CRISPR/Cas9 system utilizes a gRNA that provides targeting of a CRISPR/Cas9-based system. The gRNA is a fusion of two non-coding RNAs: crRNA and tracrRNA. The sgRNA can target any desired DNA sequence by exchanging the sequence encoding the 20bp pre-spacer sequence, which confers targeting specificity by complementary base pairing with the desired DNA target. The gRNA simulates the naturally occurring crRNA:tracrRNA duplex involved in the type II effector system. This duplex can include, for example, a 42-nucleotide crRNA and a 75-nucleotide tracrRNA, acting as a guide for Cas9 to cut the target nucleic acid.

The terms "target region", "target sequence" or "protospacer sequence", used interchangeably herein, refer to the region of the target gene targeted by a CRISPR/Cas9-based system. The CRISPR/Cas9-based system may include at least one gRNA, wherein the gRNA targets different DNA sequences. The target DNA sequences may be overlapping. The target sequence or protospacer sequence is followed by a PAM sequence at the 3' end of the protospacer sequence. Different type II systems have different PAM requirements. For example, the S. pyogenes type II system uses an "NGG" sequence, where "N" can be any nucleotide.

In certain embodiments, the number of gRNAs administered to the cells can be at least 1 gRNA, at least 2 different gRNAs, at least 3 different gRNAs, at least 4 different gRNAs, at least 5 different gRNAs, at least 6 different gRNAs, at least 7 different gRNAs, at least 8 different gRNAs, at least 9 different gRNAs, at least 10 different gRNAs, at least 11 different gRNAs, at least 12 different gRNAs, at least 13 different gRNAs, at least 14 different gRNAs, at least 15 different gRNAs, at least 16 different gRNAs, at least 17 different gRNAs, at least 18 different gRNAs, at least 19 different gRNAs, at least 20 different gRNAs, at least 25 different gRNAs, at least 30 different gRNAs, at least 35 different gRNAs, at least 40 different gRNAs, at least 45 different gRNAs, or at least 50 different gRNAs.

In certain embodiments, the number of gRNAs administered to a cell can be from at least 1 gRNA to at least 50 different gRNAs, at least 1 gRNA to at least 45 different gRNAs, at least 1 gRNA to at least 40 different gRNAs, at least 1 gRNA to at least 35 different gRNAs, at least 1 gRNA to at least 30 different gRNAs, at least 1 gRNA to at least 25 different gRNAs, at least 1 gRNA to at least 20 different gRNAs, at least 1 gRNA to at least 16 different gRNAs, at least 1 gRNA to at least 12 different gRNAs, at least 1 gRNA to at least 8 different gRNAs, at least 1 gRNA to at least 4 different gRNAs, at least 4 gRNAs to at least 50 different gRNAs, at least 4 different gRNAs to at least 45 different gRNAs, at least 4 different gRNAs to at least 40 different gRNAs, at least 4 different gRNAs to at least 3 ...5 different gRNAs, at least 4 different gRNAs to at least 40 different gRNAs, at least 4 different gRNAs to at least 35 different gRNAs, at least 4 different gRNAs to at least 30 different gRNAs, at least 1 gRNA to at least 25 different gRNAs, at least 1 gRNA to at least 20 different gRNAs, at least 1 gRNA to at least 16 different gRNAs, at least 1 gRNA to at least 12 different gRNAs, at least 1 gRNA to at least 8 different gRNAs, at least 1 gRNA to at least 4 different gRNAs different gRNAs to at least 30 different gRNAs, at least 4 different gRNAs to at least 25 different gRNAs, at least 4 different gRNAs to at least 20 different gRNAs, at least 4 different gRNAs to at least 16 different gRNAs, at least 4 different gRNAs to at least 12 different gRNAs, at least 4 different gRNAs to at least 8 different gRNAs, at least 8 different gRNAs to at least 50 different gRNAs, at least 8 different gRNAs to at least 45 different gRNAs, at least 8 different gRNAs to at least 40 different gRNAs, at least 8 different gRNAs to at least 35 different gRNAs, 8 different gRNAs to at least 30 different gRNAs, at least 8 different gRNAs to at least 25 different gRNAs, 8 different gRNAs to at least 20 different gRNAs, at least 8 different gRNAs to at least 16 different gRNAs, or 8 different gRNAs to at least 12 different gRNAs.

In some embodiments, gRNA is selected to increase or decrease the transcription of target gene. In some embodiments, the region of the transcription start site (TSS) upstream of the gRNA targeting target gene (such as PCSK9), such as the region between 0-1000bp upstream of the transcription start site of the target gene. In some embodiments, the region between 0-50bp, 0-100bp, 0-150bp, 0-200bp, 0-250bp, 0-300bp, 0-350bp, 0-400bp, 0-450bp, 0-500bp, 0-550bp, 0-600bp, 0-650bp, 0-700bp, 0-750bp, 0-800bp, 0-850bp, 0-900bp, 0-950bp or 0-1000bp upstream of the transcription start site of the gRNA targeting target gene. In certain embodiments, the gRNA targets the region within about 100bp, about 200bp, about 300bp, about 400bp, about 500bp, about 600bp, about 700bp, about 800bp, about 900bp, about 1000bp, about 1100bp, about 1200bp, about 1300bp, about 1400bp or about 1500bp upstream of the transcription start site of the target gene. In one embodiment, the gRNA targets the region 0-300bp upstream of the TSS of the target gene.

In some embodiments, the region downstream of the transcription start site of the gRNA targeting target gene, such as the region between 0-1000bp downstream of the transcription start site of the target gene. In some embodiments, the region between 0-50bp, 0-100bp, 0-150bp, 0-200bp, 0-250bp, 0-300bp, 0-350bp, 0-400bp, 0-450bp, 0-500bp, 0-550bp, 0-600bp, 0-650bp, 0-700bp, 0-750bp, 0-800bp, 0-850bp, 0-900bp, 0-950bp or 0-1000bp downstream of the transcription start site of the gRNA targeting target gene. In certain embodiments, the gRNA targets a region within about 100bp, about 200bp, about 300bp, about 400bp, about 500bp, about 600bp, about 700bp, about 800bp, about 900bp, about 1000bp, about 1100bp, about 1200bp, about 1300bp, about 1400bp or about 1500bp downstream of the transcription start site of the target gene. In one embodiment, the gRNA targets a region 0-300bp downstream of the TSS of the target gene.

Proprotein convertase subtilisin/Kexin type 9 (PCSK9) may also be referred to as subtilisin/Kexin-like protease PC9. Human PCSK9 has a cytogenetic location of 1p32.3, and genomic coordinates are at positions 55,039,548-55,064,852 on the forward strand of chromosome 1. BSND is a gene upstream of PCSK9 on the forward strand. The NCBI gene ID of human PCSK9 is 255738, the Ref Seq accession number is NM_174936.4, the Ref Seq accession number is NP_777596.2, and the Ensembl gene ID is ENSG00000169174.

The genomic location of mouse PCSK9 is 4,4C7, and has a genomic sequence of chromosome 4 at position NC_000070.07. BSND is a gene upstream of mouse PCSK9 on the forward strand. The NCBI gene ID of mouse PCSK9 is 100102, the Ref Seq accession number is NM_153565.2, the Ref Seq accession number is NP_705793.1, and the Ensembl gene ID is ENSMUSG00000044254.

The genomic location of rhesus monkey (Macaca mulatta) PCSK9 is NC_041754.1. ENSMMUG0000005740 is the gene upstream of monkey PCSK9 on the forward strand. The Ref Seq accession number of monkey PCSK9 is NM_001112660.1, the Ref Seq accession number is NP_001106130.1, and the Ensembl gene ID is ENSMMUG00000005736.

The present disclosure provides sgRNA sequences targeting mouse PCSK9 target genes. Exemplary sgRNAs include, but are not limited to, those listed in Table 3. The present disclosure also provides sgRNA sequences targeting human PCSK9. Exemplary sgRNAs include, but are not limited to, those listed in Table 4. The present disclosure also provides sgRNA sequences targeting monkey PCSK9. Exemplary sgRNAs include, but are not limited to, those listed in Table 5.

Table 3. Exemplary mouse PCSK9 sgRNA sequences

describe sequence SEQ ID NO: Mouse PCSK9 sgRNA1 TGGACGCGCAGGCTGCCGGT SEQ ID NO: 27 Mouse PCSK9 sgRNA2 CCACCTTCACGTGGACGCGC SEQ ID NO: 28 Mouse PCSK9 sgRNA3 GTGGACGCGCAGGCTGCCGG SEQ ID NO: 29 Mouse PCSK9 sgRNA4 CTCTCTCTTTCTGAGGCTAG SEQ ID NO: 30 Mouse PCSK9 sgRNA5 CACGTGGACGCGCAGGCTGC SEQ ID NO: 31 Mouse PCSK9 sgRNA6 TTAAGAGGGGGGAATGTAAC SEQ ID NO: 32 Mouse PCSK9 sgRNA7 AACCTGATCCTTTAGTACCG SEQ ID NO: 33 Mouse PCSK9 sgRNA8 TCAGAGAGGATCTTCCGATG SEQ ID NO: 34 Mouse PCSK9 sgRNA9 GGATCTTCCGATGGGGCTCG SEQ ID NO: 35 Mouse PCSK9 sgRNA10 GCGTCATTTGACGCTGTCTG SEQ ID NO: 36 Mouse PCSK9 sgRNA11 TCATTTGACGCTGTCTGGGG SEQ ID NO: 37 Mouse PCSK9 sgRNA12 GATCCTTTAGTACCGGGGCC SEQ ID NO: 38 Mouse PCSK9 sgRNA13 TGCAGCCCAATTAGGATTTG SEQ ID NO: 39

Table 4. Exemplary human PCSK9 sgRNA sequences

Table 5. Exemplary monkey PCSK9 sgRNA sequences

In one embodiment, the gRNA targets the promoter region of the target gene. In one embodiment, the gRNA targets the enhancer region of the target gene. gRNA can be divided into a target binding region, a Cas9 binding region, and a transcription termination region. The target binding region hybridizes with the target region in the target gene. The method for designing such a target binding region is known in the art, see, for example, Doench et al., Nat Biotechnol. (2014) 32: 1262-7 and Doench et al., Nat Biotechnol. (2016) 34: 184-91, which are incorporated herein by reference in their entirety. Design tools can be obtained at, for example, the target Finder of Feng Zhang Laboratory, the Target Finder (E-CRISP) of Michael Boutros Laboratory, RGEN Tools (Cas-OFFinder), CasFinder, and CRISPR Optimal Target Finder. In certain embodiments, the target binding region can be between about 15 and about 50 nucleotides in length (about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or about 50 nucleotides in length). In certain embodiments, the target binding region can be between about 19 and about 21 nucleotides in length. In one embodiment, the target binding region is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.

In one embodiment, the target binding region is complementary to the target region in the target gene, such as fully complementary. In one embodiment, the target binding region is substantially complementary to the target region in the target gene. In one embodiment, the target binding region comprises no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides that are not complementary to the target region in the target gene.

In one embodiment, the target binding region is engineered to improve stability or extend half-life, for example, by incorporating non-natural nucleotides or modified nucleotides into the target binding region, by removing or modifying RNA destabilizing sequence elements, by adding RNA stabilizing sequence elements, or by increasing the stability of the Cas9/gRNA complex. In one embodiment, the target binding region is engineered to enhance its transcription. In one embodiment, the target binding region is engineered to reduce the formation of secondary structure. In one embodiment, the Cas9 binding region of the gRNA is modified to enhance the transcription of the gRNA. In one embodiment, the Cas9 binding region of the gRNA is modified to improve the stability or assembly of the Cas9/gRNA complex.

Delivery System

The present disclosure also provides delivery systems for introducing components of the systems and compositions herein into cells, tissues, organs or organisms.The delivery system can include one or more delivery vehicles and/or carriers.

Cargo

The delivery system may include one or more carriers. The carrier may include one or more components of the systems and compositions herein. The carrier may include one or more of the following: i) a plasmid encoding one or more Cas proteins; ii) a plasmid encoding one or more guide RNAs, iii) mRNAs of one or more Cas proteins; iv) one or more guide RNAs; v) one or more Cas proteins; vi) any combination thereof. In some instances, the carrier may include a plasmid encoding one or more Cas proteins and one or more (e.g., multiple) guide RNAs. In some embodiments, the carrier may include mRNAs encoding one or more Cas proteins and one or more guide RNAs.

In some instances, the carrier may include one or more Cas proteins and one or more guide RNAs, for example, in the form of a ribonucleoprotein complex (RNP). The ribonucleoprotein complex may be delivered by the methods and systems herein. In some cases, the ribonucleoprotein may be delivered using a shuttle agent based on a polypeptide. In one example, the ribonucleoprotein may be delivered using a synthetic peptide comprising an endosome leakage domain (ELD) operably connected to a cell penetration domain (CPD), a histidine-rich domain and a CPD, such as described in WO2016161516.

Physical delivery

In certain embodiments, the cargo can be introduced into the cell by a physical delivery method. Examples of physical methods include microinjection, electroporation, and hydrodynamic delivery.

Microinjection

Microinjection of cargo directly into cells can achieve high efficiencies, e.g., greater than 90% or about 100%. In certain embodiments, microinjection can be performed using a microscope and a needle (e.g., 0.5-5.0 μm in diameter) to pierce the cell membrane and deliver the cargo directly to a target site within the cell. Microinjection can be used for in vitro and ex vivo delivery.

Plasmids containing sequences encoding Cas proteins and/or guide RNAs, mRNAs and/or guide RNAs can be microinjected. In some cases, microinjection can be used to i) deliver DNA directly to the nucleus, and/or ii) deliver mRNA (e.g., in vitro transcribed) to the nucleus or cytoplasm. In some instances, microinjection can be used to deliver sgRNA directly to the nucleus, and deliver mRNA encoding Cas to the cytoplasm, such as to promote translation of Cas and shuttling to the nucleus.

Microinjection can be used to generate genetically modified animals. For example, gene editing vectors can be injected into zygotes to allow efficient germline modification. Such methods can produce normal embryos and full-term mouse pups with the desired modifications. Microinjection can also be used to provide transient upregulation or downregulation of specific genes within the genome of a cell, such as using CRISPRa and CRISPRi.

Electroporation

In some embodiments, the carrier and/or delivery vehicle can be delivered by electroporation. Electroporation can use pulsed high voltage current to instantaneously open nanometer-sized holes in the cell membrane of cells suspended in a buffer, allowing components with a hydrodynamic diameter of tens of nanometers to flow into the cell. In some cases, electroporation can be used for various cell types and the carrier is efficiently transferred into the cell. Electroporation can be used for in vitro and ex vivo delivery.

Electroporation can also be used to deliver cargo to the nucleus of mammalian cells by applying specific voltages and reagents, such as by nuclear transfection. Such methods include those described in the following documents: Wu Y, et al. (2015). Cell Res 25: 67-79; Ye L, et al. (2014). Proc Natl Acad Sci USA 111: 9591-6; Choi PS, Meyerson M. (2014). Nat Commun 5: 3728; Wang J, Quake SR. (2014). Proc Natl Acad Sci 111: 13157-62. Electroporation can also be used to deliver cargo in vivo, for example using the method described in Zuckermann M, et al. (2015). Nat Commun 6: 7391.

Hydrodynamic delivery

Hydrodynamic delivery can also be used to deliver carriers, such as for in vivo delivery. In some instances, hydrodynamic delivery can be performed by rapidly pushing a large volume (8-10% body weight) solution containing a gene editing carrier into the bloodstream of a subject (e.g., an animal or human), such as through the tail vein for mice. Since blood is incompressible, a large amount of liquid may cause an increase in hydrodynamic pressure, thereby temporarily enhancing the permeability to endothelial cells and parenchymal cells, allowing carriers that normally cannot pass through the cell membrane to enter the cell. This method can be used to deliver naked DNA plasmids and proteins. The delivered carrier may be enriched in the liver, kidneys, lungs, muscles, and/or heart.

Transfection

The cargo, such as nucleic acid, can be introduced into the cell by a transfection method for introducing nucleic acid into the cell. Examples of transfection methods include calcium phosphate-mediated transfection, cationic transfection, liposome transfection, dendrimer transfection, heat shock transfection, magnetofection, liposome transfection, impalefection, photofection, proprietary reagents to enhance nucleic acid uptake.

Delivery vehicle

The delivery system may include one or more delivery vehicles. The delivery vehicle may deliver the vehicle to a cell, tissue, organ or organism (e.g., animal or plant). The vehicle may be packaged, carried or otherwise associated with a delivery vehicle. The delivery vehicle may be selected according to the type of vehicle to be delivered and/or whether the delivery is in vitro and/or in vivo. Examples of delivery vehicles include vectors, viruses, non-viral vehicles and other delivery agents described herein.

According to the delivery vehicle of the present disclosure, the maximum dimension (e.g., diameter) of less than 100 microns (μm) can be provided. In certain embodiments, the maximum dimension of the delivery vehicle is less than 10 μm. In certain embodiments, the delivery vehicle can have a maximum dimension less than 2000 nanometers (nm). In certain embodiments, the delivery vehicle can have a maximum dimension less than 1000 nanometers (nm). In certain embodiments, the delivery vehicle can have a maximum dimension less than 900nm, less than 800nm, less than 700nm, less than 600nm, less than 500nm, less than 400nm, less than 300nm, less than 200nm, less than 150nm or less than 100nm, less than 50nm. In certain embodiments, the delivery vehicle can have a maximum dimension in the range between 25nm and 200nm.

In certain embodiments, the delivery vehicle can be or include particles. For example, the delivery vehicle can be or include nanoparticles (e.g., particles having a maximum dimension (e.g., diameter) of no more than 1000 nm). The particles can be provided in different forms, such as solid particles (e.g., metals such as silver, gold, iron, titanium, non-metals, lipid-based solids, polymers), suspensions of particles, or combinations thereof. Metals, dielectrics, and semiconductor particles and hybrid structures (e.g., core-shell particles) can be prepared.

Carrier

The system, composition and/or delivery system may include one or more vectors. The present disclosure also includes a vector system. The vector system may include one or more vectors. In some embodiments, a vector refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. The vector includes a single-stranded, double-stranded or partially double-stranded nucleic acid molecule, a nucleic acid molecule comprising one or more free ends, no free ends (e.g., circular), a nucleic acid molecule comprising DNA, RNA or both, and other various polynucleotides known in the art. The vector may be a plasmid, such as a circular double-stranded DNA loop that can be inserted into an additional DNA segment, such as by standard molecular cloning techniques. Some vectors may be able to replicate autonomously in the host cell into which they are introduced (e.g., a bacterial vector and an additional mammalian vector with a bacterial replication origin). Some vectors (e.g., a non-additional mammalian vector) are integrated into the genome of the host cell after being introduced into the host cell, thereby replicating together with the host genome. In some instances, the vector may be an expression vector, such as being able to direct the expression of a gene operably connected thereto. In some cases, an expression vector may be used for expression in a eukaryotic cell. Common expression vectors useful in recombinant DNA technology generally take the form of a plasmid.

Examples of vectors include pGEX, pMAL, pRIT5, E. coli expression vectors (e.g., pTrc, pETlld), yeast expression vectors (e.g., pYepSecl, pMFa, pJRY88, pYES2, and picZ), baculovirus vectors (e.g., for expression in insect cells such as SF9 cells) (e.g., pAc series and pVL series), mammalian expression vectors (e.g., pCDM8 and pMT2PC).

The vector may comprise i) a Cas coding sequence, and/or ii) a single or at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 32, at least 48, at least 50 guide RNA coding sequences. In a single vector, each RNA coding sequence may have a promoter. Alternatively or in addition, in a single vector, there may be promoters that control (e.g., drive transcription and/or expression) multiple RNA coding sequences.

Regulatory elements

The vector may include one or more regulatory elements. The regulatory element may be operably connected to the coding sequence of the Cas protein, auxiliary protein, guide RNA (e.g., single guide RNA, crRNA and/or tracrRNA) or a combination thereof. The term "operably connected" means that the nucleotide sequence of interest is connected to the regulatory element in a manner that allows the expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into a host cell). In some instances, the vector may include: a first regulatory element operably connected to a nucleotide sequence encoding a Cas protein, and a second regulatory element operably connected to a nucleotide sequence encoding a guide RNA.

Examples of regulatory elements include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif (1990). Regulatory elements include those that direct the constitutive expression of nucleotide sequences in many types of host cells, and those that direct the expression of nucleotide sequences only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can be mainly directed to expression in desired tissues of interest such as muscle, neuron, bone, skin, blood, specific organs (e.g., liver, pancreas), or specific cell types (e.g., lymphocytes). Regulatory elements can also direct expression in a time-dependent manner, such as in a cell cycle-dependent or developmental stage-dependent manner, which may or may not be tissue or cell type specific.

Examples of promoters include one or more pol III promoters (e.g., 1, 2, 3, 4, 5 or more pol III promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5 or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5 or more pol I promoters), or a combination thereof. Examples of pol III promoters include, but are not limited to, U6 and HI promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with CMV enhancer), the SV40 promoter, the dihydrofolate reductase promoter, the actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1a promoter.

Viral vectors

The carrier can be delivered by virus. In some embodiments, a viral vector is used. The viral vector may include a virally derived DNA or RNA sequence (e.g., a retrovirus, a replication-defective retrovirus, an adenovirus, a replication-defective adenovirus, and an adeno-associated virus) for packaging into the virus. The viral vector also includes a polynucleotide carried by the virus for transfection into a host cell. Viruses and viral vectors can be used for in vitro, ex vivo, and/or in vivo delivery.

Adeno-associated virus (AAV)

The systems and compositions herein can be delivered by adeno-associated virus (AAV). AAV vectors can be used for such delivery. AAV belongs to the Parvoviridae family of the Dependovirus genus and is a single-stranded DNA virus. In some embodiments, AAV can provide a lasting source of the DNA provided, because the genomic material delivered by AAV can exist indefinitely in cells, for example, as exogenous DNA or with some modifications, and is directly integrated into the host DNA. In some embodiments, AAV does not cause any disease or is associated with any disease in humans. The virus itself can infect cells efficiently without eliciting innate or adaptive immune responses or related toxicity.

Examples of AAVs that can be used herein include AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-8, and AAV-9. The type of AAV can be selected according to the cells to be targeted; for example, AAV serotypes 1, 2, 5 or hybrid capsids AAV1, AAV2, AAV5, or any combination thereof can be selected for targeting brain or neuronal cells; and AAV4 can be selected for targeting cardiac tissue. AAV8 can be used for delivery to the liver. AAV-2-based vectors were initially proposed for CFTR delivery to CF airways, and other serotypes such as AAV-1, AAV-5, AAV-6, and AAV-9 showed improved gene transfer efficiency in various lung epithelial models. Examples of cell types targeted by AAV are described in Grimm, D. et al, J. Virol. 82: 5887-5911 (2008) and WO2021/183807A1, which are incorporated herein by reference in their entirety.

CRISPR-Cas AAV particles can be produced in HEK 293T cells. Once particles with specific tropism are produced, they can be used to infect target cell lines in essentially the same way as natural viral particles. This may allow CRISPR-Cas components to persist in infected cell types, which is also the reason why this delivery version is particularly suitable for situations where long-term expression is required. Examples of doses and formulations of AAV that can be used include those described in U.S. Patent Nos. 8,454,972 and 8,404,658.

A variety of strategies can be used to deliver the systems and compositions herein using AAV. In some instances, the coding sequences of Cas and gRNA can be packaged directly into a DNA plasmid vector and delivered via an AAV particle. In some instances, AAV can be used to deliver gRNA to cells that have been previously engineered to express Cas. In some instances, the coding sequences of Cas and gRNA can be manufactured into two separate AAV particles for co-transfection of target cells. In some instances, markers, tags, and other sequences can be packaged in the same AAV particles as the coding sequences of Cas and/or gRNA.

Lentivirus

The systems and compositions herein can be delivered via lentivirus. Lentiviral vectors can be used for such delivery. Lentiviruses are complex retroviruses that have the ability to infect and express their genes in mitotic and post-mitotic cells.

Examples of lentiviruses include human immunodeficiency virus (HIV), which can target a wide range of cell types using envelope glycoproteins of other viruses; minimal non-primate lentiviral vectors based on equine infectious anemia virus (EIAV), which can be used for ocular therapy. In certain embodiments, a self-inactivating lentiviral vector with siRNA targeting common exons shared by HIV tat/rev, a TAR decoy localized to the nucleolus, and an anti-CCR5 specific hammerhead ribozyme (see, e.g., DiGiusto et al. (2010) Sci Transl Med 2:36ra43) can be used and/or adapted for use in the nucleic acid targeting system herein.

Lentiviruses can be pseudotyped with other viral proteins, such as the G protein of vesicular stomatitis virus. In doing so, the cellular tropism of the lentivirus can be altered to be as broad or narrow as desired. In some cases, to increase safety, second- and third-generation lentiviral systems may separate essential genes into three plasmids, which may reduce the likelihood of accidental reconstitution of live viral particles within cells.

In certain examples, using the ability to integrate, lentiviruses can be used to create libraries containing various genetically modified cells, for example, for screening and/or studying genes and signaling pathways.

Adenovirus

The systems and compositions herein can be delivered by adenovirus. Adenoviral vectors can be used for such delivery. Adenoviruses include non-enveloped viruses with icosahedral nucleocapsids containing double-stranded DNA genomes. Adenoviruses can infect dividing cells and non-dividing cells. In certain embodiments, adenoviruses are not integrated into the genome of host cells, which can be used to limit the off-target effects of CRISPR-Cas systems in gene editing applications.

Non-viral vectors

The delivery vehicle may include a non-viral vehicle. Generally, methods and vehicles capable of delivering nucleic acids and/or proteins can be used to deliver the systems and compositions herein. Examples of non-viral vehicles include lipid nanoparticles, cell penetrating peptides (CPPs), DNA nanowires, gold nanoparticles, streptolysin O, multifunctional coated nanodevices (MENDs), lipid-coated mesoporous silica particles, and other inorganic nanoparticles.

Lipid particles

The delivery vehicle may include lipid particles, such as lipid nanoparticles (LNPs) and liposomes.

Lipid Nanoparticles (LNP)

LNP can be encapsulated in cationic lipid particles (e.g., liposomes) by nucleic acid, and can be delivered to cells relatively easily. In some instances, lipid nanoparticles do not contain any viral components, which helps to minimize safety and immunogenicity. Lipid particles can be used for external, in vitro and in vivo delivery. Lipid particles can be used for cell colonies of various sizes.

In some instances, LNP can be used to deliver DNA molecules (e.g., those comprising coding sequences of Cas and/or gRNA) and/or RNA molecules (e.g., mRNA, gRNA of Cas). In some cases, LNP can be used to deliver the RNP complex of Cas/gRNA.

In certain embodiments, LNPs are used to deliver mRNA and gRNA, such as an mRNA fusion molecule comprising DNMT3A-DNMT3L (3A-3L)-dCas9-KRAB and at least one sgRNA targeting PCSK9.

The components of LNPs may include cationic lipids 1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP), 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA), 1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA), (3-o-[2-(methoxypolyethyleneglycol 2000)succinoyl]-1,2-dimyristoyl-sn-glycol (PEG-S-DMG), R-3-[(ro-methoxy-poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-C-DOMG), and any combination thereof. The preparation and encapsulation of LNPs may be adapted from Conway et al. al, Molecular Therapy, vol.27, no.4, pages 866-877, Apr.2019 and Rosin et al, Molecular Therapy, vol.19, no.12, pages 1286-2200, Dec.201.

In certain embodiments, LNP may include ionizable lipids. In certain embodiments, ionizable lipids include but are not limited to pH responsive ionizable lipids, thermal responsive ionizable lipids and light responsive ionizable lipids. In certain embodiments, ionizable lipids include cationic lipids and anionic lipids ionized under certain conditions such as but not limited to pH, temperature or light. In certain embodiments, the molar ratio of the ionizable lipids of the LNP is 20% to about 70% (e.g., about 20% to about 70%, about 20% to about 65%, about 20% to about 60%, about 20% to about 55%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 20% to about 25%, about 30% to about 70%, about 30% to about 65%, about 30% to about 60%, about 3 In some embodiments, the molar ratio of ionizable lipids of the LNPs is about 45% to about 50%. In some embodiments, the molar ratio of ionizable lipids of the LNPs is about 45% to about 50%. In some embodiments, the molar ratio of ionizable lipids of the LNPs is about 45% to about 50%.

In some embodiments, LNP can comprise PEGylated lipid.In some embodiments, the mol ratio of the PEGylated lipid of described LNP is 0% to about 30% (for example, about 0% to about 30%, about 0% to about 25%, about 0% to about 20%, about 0% to about 15%, about 0% to about 10%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 15%, about 20% to about 30% or about 20% to about 25%).In some embodiments, the mol ratio of the PEGylated lipid of described LNP is about 1%.

In some embodiments, the LNP may include a supporting lipid. In some embodiments, the molar ratio of the supporting lipid of the LNP is 5% to about 50% (e.g., about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 15% to about 10%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 1 In some embodiments, the molar ratio of the supporting lipid of the LNP is about 9%.

In some embodiments, LNP can include cholesterol.In some embodiments, the mol ratio of the cholesterol of the LNP is 10% to about 50% (for example, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 15%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 20% to about 25%, about 30% to about 50%, about 30% to about 45%, about 30% to about 40%, about 30% to about 35%, about 40% to about 50% or about 40% to about 45%). In some embodiments, the mol ratio of the cholesterol of the LNP is about 40% to about 45%.

In some embodiments, LNP can comprise a mixture of ionizable lipid (20%-70%, mol ratio), PEGylated lipid (0%-30%, mol ratio), supporting lipid (30%-50%, mol ratio) and cholesterol (10%-50%, mol ratio). In some embodiments, the LNP can comprise a mixture of ionizable lipid (45-50%, mol ratio), PEGylated lipid (1%, mol ratio), supporting lipid (9%, mol ratio) and cholesterol (40-50%, mol ratio).

Liposomes

In some embodiments, lipid granule can be liposome.Liposome is the spherical vesicle structure that is composed of single layer or multilayer lipid bilayer around internal aqueous compartment and relatively impermeable external lipophilic phospholipid bilayer.In some embodiments, liposome is biocompatible, nontoxic, can deliver both hydrophilic and lipophilic drug molecules, protect their carrier from being degraded by cytoplasmic enzymes, and their load is transported through biomembrane and blood-brain barrier (BBB).

Liposomes can be made from several different types of lipids, such as phospholipids. Liposomes can include natural phospholipids and lipids such as 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), sphingomyelin, egg phosphatidylcholine, monosialoganglioside, or any combination thereof.

Several other additives can be added to the liposomes in order to modify their structure and properties. For example, the liposomes can further comprise cholesterol, sphingomyelin and/or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), for example to improve stability and/or prevent leakage of cargo inside the liposomes.

Stable Nucleic Acid Lipid Particles (SNALP)

In some embodiments, the lipid granule can be a stable nucleic acid lipid granule (SNALP). SNALP can include ionizable lipids (DLinDMA) (e.g., cations at low pH), neutral auxiliary lipids, cholesterol, diffusible polyethylene glycol (PEG) lipids or any combination thereof. In some instances, SNALP can include synthetic cholesterol, dipalmitoylphosphatidylcholine, 3-N-[(w-methoxypolyethylene glycol) 2000] carbamoyl]-1,2-dimyristyloxypropylamine and cation 1,2-dilinoleyloxy-3-N, N-dimethylaminopropane. In some instances, SNALP can include synthetic cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine, PEG-cDMA and 1,2-dilinoleyloxy-3-(N, N-dimethyl) aminopropane (DLinDMA).

Other lipids

The lipid particles may also include one or more other types of lipids, for example, cationic lipids such as amino lipids 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), DLin-KC2-DMA4, Cl2-200 and auxiliary lipids distearoylphosphatidylcholine, cholesterol and PEG-DMG.

Lipoplexes and/or polyplexes

In certain embodiments, the delivery vehicle comprises a lipid complex and/or a polymer complex. The lipid complex can bind to a negatively charged cell membrane and induce endocytosis. Examples of cationic lipid complexes can be complexes comprising lipids and non-lipid components. Examples of lipid complexes and polymer complexes include FuGENE-6 reagents, non-lipid solutions containing lipids and other components, zwitterionic amino lipids (ZAL), Ca2p (e.g., forming DNA/Ca2 ⁺ microcomplexes), polyethyleneimine (PEI) (e.g., branched PEI) and poly (L-lysine) (PLL).

Cell Penetrating Peptides

In certain embodiments, the delivery vehicle comprises a cell penetrating peptide (CPP). CPPs are short peptides that facilitate cellular uptake of a variety of molecular cargoes, such as from nanoparticles to small chemical molecules and large DNA fragments.

CPPs can have different sizes, amino acid sequences and charges. In some instances, CPPs can translocate the plasma membrane and facilitate the delivery of various molecular cargoes to the cytoplasm or organelles. CPPs can be introduced into cells by different mechanisms, such as direct penetration into the membrane, endocytosis-mediated entry, and translocation by forming temporary structures.

CPP can have the amino acid composition of positively charged amino acid such as lysine or arginine containing high relative abundance, or have the sequence of alternating pattern containing polar/charged amino acid and non-polar hydrophobic amino acid.These two types of structures are respectively referred to as polycationic or amphipathic.The third type of CPP is hydrophobic peptide, containing only non-polar residues with low net charge or having hydrophobic amino acid groups that are crucial for cellular uptake.Another type of CPP is the trans-activating transcription activator (Tat) from human immunodeficiency virus I (HIV-I).Examples of CPP include penetratin, Tat (48-60), transmembrane peptide (Transportan) and (R-AhX-R4) (AhX refers to aminocaproyl).Examples of CPP and related applications also include those described in U.S. Patent No. 8,372,951.

CPPs can be used fairly easily for in vitro and ex vivo work, and often require extensive optimization for each cargo and cell type. In some instances, CPPs can be covalently linked directly to Cas proteins, then complexed with gRNA and delivered to cells. In some instances, CPP-Cas and CPP-gRNA can be delivered separately to multiple cells. CPPs can also be used to deliver RNPs.

DNA nanowires

In certain embodiments, the delivery vehicle comprises a DNA nanocluster. A DNA nanocluster refers to a spherical structure of DNA (e.g., having the shape of a yarn ball). The nanocluster can be synthesized by rolling circle amplification using a palindromic sequence that facilitates self-assembly of the structure. The ball can then be loaded with a payload. Examples of DNA nanocluster are described in Sun Wet al, J Am Chem Soc. 2014 Oct 22; 136(42): 14722-5; and Sun Wet al, Angew Chem Int Ed Engl. 2015 Oct 5; 54(41): 12029-33. The DNA nanocluster may have a palindromic sequence that is complementary to a portion of the gRNA within the Cas:gRNA ribonucleoprotein complex. The DNA nanocluster can be coated, for example, with PEI, to induce endosomal escape.

Gold Nanoparticles

In certain embodiments, the delivery vehicle includes gold nanoparticles (also referred to as AuNP or colloidal gold). Gold nanoparticles can form complexes with carriers such as Cas:gRNA RNP. Gold nanoparticles can be coated, for example, coated in silicates and endosomal disruptive polymers PAsp (DET). Examples of gold nanoparticles include spherical nucleic acids (SNA ^™ ) constructs of AuraSenseTherapeutics, and those described in Mout R, et al. (2017). ACS Nano 11: 2452-8; Lee K, et al. (2017). Nat Biomed Eng 1: 889-901.

iTOP

In certain embodiments, the delivery vehicle includes iTOP. iTOP refers to a combination of small molecules that drive efficient intracellular delivery of natural proteins independently of any transduction peptide. iTOP can be used for transduction induced by cell osmosis and propane betaine, using NaCl-mediated hyperosmoticity together with transduction compounds (propane betaine) to trigger macropinocytotic uptake of extracellular macromolecules into cells. Examples of iTOP methods and reagents include those described in D'Astolfo DS, Pagliero RJ, Pras A, et al. (2015). Cell 161: 674-690.

Polymer-based particles

In some embodiments, the delivery vehicle may include polymer-based particles (e.g., nanoparticles). In some embodiments, the polymer-based particles may simulate the membrane fusion mechanism of the virus. The polymer-based particles may be synthetic copies of the influenza virus mechanism and may form transfection complexes with various types of nucleic acids (siRNA, miRNA, plasmid DNA or shRNA, mRNA) that are taken up by cells through the endocytic pathway (a process involving the formation of acidic compartments). The low pH in the late endosome acts as a chemical switch, making the surface of the particles hydrophobic and facilitating membrane penetration. Once entering the cytosol, the particles release their payload for cellular action. This active endosome escape (Active EndosomeEscape) technology is safe and maximizes transfection efficiency because it uses a natural uptake pathway. In some embodiments, the polymer-based particles may include alkylated and carboxyalkylated branched polyethyleneimine. In some instances, the polymer-based particles are VIROMER, such as VIROMER RNAi, VIROMER RED, VIROMER mRNA, VIROMER CRISPR. Examples of methods of delivering the systems and compositions herein include those described in: Bawage SS et al., Synthetic mRNA expressed Cas13amitigates RNAvirus infections, www.biorxiv.org/content/10.1 101/370460v1.full doi:doi.org/10.1101/370460, RED, a powerful tool for transfection of keratinocytes.doi:10.13140/RG.2.2.16993.61281, Transfection-Factbook2018:technology,product overview,users'data.,doi:10.13140/RG.2.2.23912.16642.

Streptolysin O (SLO)

The delivery vehicle can be streptolysin O (SLO). SLO is a toxin produced by group A streptococci that acts by creating pores in mammalian cell membranes. SLO can act in a reversible manner, which allows proteins (e.g., up to 100 kDa) to be delivered to the cytosol of cells without compromising overall viability. Examples of SLO include those described in the following literature: Sierig G, et al. (2003). Infect Immun 71: 446-55; Walev I, et al. (2001). Proc Natl Acad Sci USA 98: 3185-90; Teng KW, et al. (2017). Elife 6: e25460. Multifunctional Encapsulated Nanodevice (MEND)

The delivery vehicle may include a multifunctional membrane-encapsulated nanodevice (MEND). MEND may include condensed plasmid DNA, a PLL core, and a lipid membrane shell. MEND may further include a cell penetrating peptide (e.g., stearoyl octaarginine). The cell penetrating peptide may be in a lipid shell. The lipid envelope may be modified with one or more functional components, such as one or more of the following: polyethylene glycol (e.g., to increase vascular circulation time), ligands for targeting specific tissues/cells, other cell penetrating peptides (e.g., for larger cell delivery), lipids that enhance endosomal escape, and nuclear delivery tags. In some instances, the MEND may be a four-layer MEND (T-MEND), which may target the nucleus and mitochondria. Examples of MEND include those described in the following documents: Kogure K, et al. (2004). J Control Release 98: 317-23; Nakamura T, et al. (2012). Ace Chem Res 45: 1113-21.

Lipid-coated mesoporous silica particles

The delivery vehicle may include lipid-coated mesoporous silica particles. The lipid-coated mesoporous silica particles may include a mesoporous silica nanoparticle core and a lipid membrane shell. The silica core may have a large internal surface area, resulting in a high cargo loading capacity. In certain embodiments, the pore size, pore chemistry, and total particle size may be modified to load different types of cargoes. The lipid envelope of the particles may also be modified to maximize cargo loading, increase circulation time, and provide precise targeting and cargo release. Examples of lipid-coated mesoporous silica particles include those described in the following literature: Du X, et al. (2014). Biomaterials 35: 5580-90; Durfee PN, et al. (2016). ACS Nano 10: 8325-45.

Inorganic Nanoparticles

The delivery vehicle may include inorganic nanoparticles. Examples of inorganic nanoparticles include carbon nanotubes (CNTs) (e.g., described in Bates K and Kostarelos K. (2013). Adv Drug Deliv Rev 65: 2023-33), bare mesoporous silica nanoparticles (MSNPs) (e.g., described in Luo GF, et al. (2014). Sci Rep 4: 6064), and dense silica nanoparticles (SiNPs) (e.g., described in Luo D and Saltzman WM. (2000). Nat Biotechnol 18: 893-5).

Instructions

The compositions and systems herein can be used for various applications, including modification of non-animal organisms such as plants and fungi and modification of animals, treatment and diagnosis of diseases in plants, animals and humans. Generally, the compositions and systems can be introduced into cells, tissues, organs or organisms, where they modify the expression and/or activity of one or more genes (e.g., PCSK9).

In certain embodiments, the expression of the PCSK9 gene product is reduced in cells into which the compositions and systems described herein are introduced. In certain embodiments, the reduction in the expression of the PCSK9 gene product is temporary. In certain embodiments, the reduction in the expression of the PCSK9 gene product is stable. In certain embodiments, the reduction in the expression of the PCSK9 gene product is heritable.

In certain embodiments, multiple cells modified by the compositions and systems described herein comprise expression of a PCSK9 gene product that is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% relative to cells to which the compositions and systems described herein have not been introduced.

In certain embodiments, cells expanded or derived from a plurality of cells modified by the compositions and systems described herein also comprise expression of a PCSK9 gene product that is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% relative to cells expanded or derived from cells that have not been introduced with the compositions and systems described herein.

Cells and Organisms

The present disclosure provides cells, tissues, and organisms comprising the engineered Cas protein, CRISPR-Cas system, polynucleotides encoding one or more components of the CRISPR-Cas system, and/or vectors comprising the polynucleotides. The present disclosure also provides nucleotide sequences encoding effector proteins in any method or composition described herein, and the nucleotide sequences are codon-optimized for expression in eukaryotic organisms or eukaryotic cells. In one embodiment of the present disclosure, the codon-optimized effector protein is any Cas protein discussed herein, and is codon-optimized for operability in eukaryotic cells or organisms, such as such cells or organisms mentioned elsewhere herein, such as, but not limited to, yeast cells or mammalian cells or organisms, including mouse cells, rat cells, and human cells or non-human eukaryotic organisms such as plants.

In certain embodiments, modification of the target locus of interest may result in: a eukaryotic cell in which the expression of at least one gene product is altered; a eukaryotic cell in which the expression of at least one gene product is altered, wherein the expression of the at least one gene product is increased; a eukaryotic cell in which the expression of at least one gene product is altered, wherein the expression of the at least one gene product is decreased; or a eukaryotic cell comprising an edited genome.

In certain embodiments, the eukaryotic cell can be a mammalian cell or a human cell.

In other embodiments, the non-naturally occurring or engineered compositions, vector systems, or delivery systems described in this specification can be used for: site-specific gene knockout; site-specific genome editing; RNA sequence-specific interference; or multiplex genome engineering.

Also provided herein is a gene product from a cell, cell line or organism described herein. In some embodiments, the amount of the gene product expressed can be greater than or less than the amount of the gene product from a cell without the expression or editing of the genome that has not been changed. In some embodiments, the gene product can be changed compared to the gene product from a cell without the expression or editing of the genome that has not been changed.

Exemplary Therapies

The present disclosure provides the use of the CRISPR-Cas system in treating various diseases and disorders. In certain embodiments, the disclosure described herein relates to a method of treatment, wherein cells are edited by CRISPR or a base editor to regulate at least one gene, and the edited cells are subsequently administered to patients in need. In certain embodiments, the editing involves knocking in, knocking out or knocking down the expression of at least one target gene in the cell. In a specific embodiment, the editing may include an exogenous gene, a small gene or a sequence of one or more exons in a trans or natural or synthetic trans manner to insert into the locus of the target gene, the hotspot locus, the safe harbor locus of the genomic position of the gene (wherein new genes or gene elements can be introduced without destroying the expression or regulation of adjacent genes), or by inserting or deleting one or more mutations in the DNA sequence encoding the regulatory element of the target gene for correction. In certain embodiments, the editing includes introducing one or more point mutations in a nucleic acid (e.g., genomic DNA) in a target cell.

In certain embodiments, the treatment is directed to a disease/disorder of an organ, including liver disease, eye disease, muscle disease, heart disease, blood disease, brain disease, kidney disease, or may include treatment for autoimmune diseases, central nervous system diseases, cancer and other proliferative diseases, neurodegenerative diseases, inflammatory diseases, metabolic disorders, musculoskeletal disorders, etc.

In some embodiments, the disease is associated with high cholesterol and provides for the regulation of cholesterol (e.g., LDL). In some embodiments, the regulation is affected by modifications in the target gene PCSK9. PCSK9 is associated with diseases and disorders such as, but not limited to, abetalipoproteinemia, adenoma, atherosclerosis, atherosclerosis, cardiovascular disease, gallstones, coronary atherosclerosis, coronary heart disease, non-insulin-dependent diabetes mellitus, hypercholesterolemia, familial hypercholesterolemia, hyperinsulinemia, hyperlipidemia, familial combined hyperlipidemia, hypobetalipoproteinemia, chronic renal failure, liver disease, liver tumors, melanoma, myocardial infarction, narcolepsy, tumor metastasis, Wilms tumor, obesity, peritonitis, pseudoxanthoma elasticum, cerebrovascular accident, vascular disease, xanthomatosis, peripheral vascular disease, myocardial ischemia , dyslipidemia, impaired glucose tolerance, xanthomatosis, multigenic hypercholesterolemia, secondary liver malignancies, dementia, overweight, chronic hepatitis C, carotid atherosclerosis, type HA hyperlipoproteinemia, intracranial atherosclerosis, ischemic stroke, acute coronary syndrome, aortic calcification, cardiovascular morbidity, type Lib hyperlipoproteinemia, peripheral arterial disease, type II familial aldosteronism, familial hypobetalipoproteinemia, autosomal recessive hypercholesterolemia, autosomal dominant hypercholesterolemia 3, coronary artery disease, liver cancer, ischemic cerebrovascular accident, and atherosclerotic cardiovascular disease NOS. Epigenetic modification of the PCSK9 gene using any of the methods described herein can be used to treat, prevent, and/or alleviate the symptoms of the diseases and disorders described herein.

Dyslipidemia is a genetic disease characterized by elevated lipid levels in the blood, leading to the occurrence of arterial blockage (atherosclerosis). These lipids include plasma cholesterol, triglycerides, high-density lipoproteins or low-density lipoproteins. Dyslipidemia increases the risk of heart attack, stroke or other circulatory system problems. Current treatments include lifestyle changes such as exercise and dietary adjustments, and the use of lipid-lowering drugs such as statins. Non-statin lipid-lowering drugs include bile acid sequestrants, cholesterol absorption inhibitors, homozygous familial hypercholesterolemia drugs, fibrates, niacin, ω-3 fatty acids and/or combination products. Treatment options usually depend on specific lipid abnormalities, although different lipid abnormalities often coexist. Treatment of children is more challenging because dietary changes may be difficult to implement, and lipid-lowering therapies have not yet been proven to be effective. Using any method described herein to carry out epigenetic modification of the PCSK9 gene can be used to treat, prevent and/or alleviate the symptoms of dyslipidemia (e.g., LDL imbalance).

The activity of PCSK9 is mainly confined to the liver, and PCSK9 is associated with dyslipidemia, PCSK9-related familial hypercholesterolemia, hypercholesterolemia (familial), gastric papillary adenocarcinoma, homozygous familial hypercholesterolemia, and nasopharyngitis. PCSK9-related familial hypercholesterolemia is an (autosomal dominant) genetic disease in which the body presents dangerous blood cholesterol levels due to the lack of low-density lipoprotein cholesterol receptors. PCSK9-related familial hypercholesterolemia affects between 1 in 500 heterozygotes and 1 in 1,000,000 homozygotes in the world's population, and is more common in white South Africans, French Canadians, Lebanese Christians, and Finns. Common symptoms of PCSK9-related familial hypercholesterolemia include elevated circulating cholesterol contained in either single low-density lipoproteins or also very low-density lipoproteins. Current treatments for PCSK9-related familial hypercholesterolemia include the administration of statins to inhibit hydroxymethylglutaryl coenzyme A reductase (HMG-CoA reductase) in the liver. Another option for treating PCSK9-related familial hypercholesterolemia is ezetimibe, which inhibits the absorption of cholesterol in the intestine.

In certain embodiments, epigenetic modification of the PCSK9 gene by any of the methods described herein can be targeted to the liver, the primary site of PCSK9 activity.

Example

Example 1: Fusion molecule plasmid construction and knockdown efficiency

Two plasmids were constructed to form the "EPICAS" system (which can be used interchangeably with the "CRISPRoff" system) (Figure 1A). The "fusion molecule" or "catalytic protein" plasmid encodes dCas9, DNMT3A, DNMT3L, and KRAB peptides. The fused DNMT3A and DNMT3L (3A3L) peptides are located at the N-terminus of dCas9, and KRAB is located at the C-terminus of dCas9. Therefore, the fusion molecule has 3A3L-dCas9-KRAB from the N-terminus to the C-terminus. The "sgRNA" plasmid encodes the sgRNA sequence targeting the PCSK9 gene. Multiple sgRNAs were designed to target the region within 250bp upstream and downstream of the transcription start site (TSS) of the mouse PCSK9 gene.

Each sgRNA plasmid was co-transfected with a catalytic protein plasmid into a mouse AML12 cell line. After 72 hours, the top 10% of GFP+ and mCherry+ cells were sorted by FACS. RT-QPCR experiments were performed to assess the mRNA expression level of Pcsk9. Twelve of the 13 sgRNAs tested showed significant downregulation of Pcsk9 expression in AML12 cells. Cells transfected with sgRNA9 showed an effective knockdown of up to about 82% (Figure 1B).

Next, the combination of sgRNA9 and other individual sgRNAs was tested to determine whether the combination of more than one sgRNA could further reduce the gene expression level of Pcsk9 in AML12 cells (Figure 1C). Among the combinations tested, sgRNA7 and sgRNA9 showed the highest inhibition level together. Each combination of sgRNAs was also tested to determine whether the combination of more than one sgRNA could reduce the gene expression level of Pcsk9 in Ai9 primary hepatocytes (Figure 1D). All combinations significantly knocked down the expression level of Pcsk9. The lowest reduction was observed in cells co-transfected with sgRNA7, sgRNA8, and sgRNA9. Pcsk9 silencing lasted for at least two weeks in primary hepatocytes, and the combination of sgRNA7 and sgRNA9 showed the highest inhibition efficiency (up to 81%) for PCSK9 gene expression. In summary, this shows that the EPICAS system can be used to induce efficient and lasting silencing of the Pcsk9 gene in mouse hepatocytes.

Example 2: In vitro transcription of mRNA encoding fusion molecules

In vitro transcription and purification were used to generate mRNA corresponding to the fusion molecule or catalytic protein of the EPICAS system. First, a plasmid containing all fusion molecule elements including the 5'UTR-DNMT3A-DNMT3L-dCas9-KRAB-3'UTR-polyA expression cassette was constructed. The plasmid sequence was linearized by digestion with XbaI and BpiI restriction endonucleases (Figure 2A). An in vitro transcription reaction containing a linearized DNA template, T7 RNA polymerase, NTP, and cap analogs was performed to produce mRNA containing N1-methyl pseudouridine. After digestion of the DNA template with DNaseI, the mRNA product was purified and buffer exchanged, and the purity of the final mRNA product was assessed by capillary gel electrophoresis (Figure 2B). 100-mer sgRNA was chemically synthesized by a commercial supplier under solid phase synthesis conditions with minimal end modification. In order to test the function of the in vitro transcribed mRNA, the Snrpn-GFP reporter system was constructed in HEK293T cells (Figure 2C). The reporter system uses a synthetic methylation-sensing promoter (conserved sequence elements from the promoter of the imprinted gene Snrpn) to control the expression of GFP. The insertion of the reporter construct in the genomic locus shows the methylation status of the adjacent sequence. The above-mentioned in vitro transcribed mRNA is co-transfected into the reporter cells with the sgRNA targeting the Snrpn gene. 8 days after transfection, 25.3% of the cells in the reporter cells were GFP negative, which was significantly higher than the control group transfected with non-targeted sgRNA (Figure 2D). GFP-negative cells were sorted by FACS and cultured for 30 days. 30 days after transfection, 93.2% of the cells in the reporter system were GFP-negative, while almost no GFP-negative cells were found in the control group (Figures 2D, 2E). 70 days and 90 days after transfection, 86.1% and 87.3% of the cells in the reporter system were GFP-negative, respectively (Figure 2I). At 150 and 400 days after transfection (i.e., up to ~400 cell divisions), 92.7% and 88.1% of the cells in the reporter system were GFP negative, respectively. This indicates the persistence of epigenomic editing using the CRISPRoff system (Figure 2D). In addition, the DNA methylation level at the Snrpn locus was analyzed by bisulfite PCR assay. The methylation level of the reporter cells (GFP-OFF group) was significantly higher than that of the control cells (GFP-ON group) (Figure 2F). This result was accompanied by high CpG methylation observed at the Snrpn locus (Figures 2F, 2G, 2I). In summary, these results indicate that transient expression and mRNA scanning of the EPICAS system silences the expression of target genes over a long period of time.

Example 3: Lipid Nanoparticle Encapsulation of mRNA and sgRNA Encoding Fusion Molecules LNP Formulation and Characteristics

LNPs were formulated using standard methods known in the art for delivery of fusion molecule mRNA and sgRNA to human hepatocytes. For mouse studies, LNPs were formulated as described previously with some modifications (1). Briefly, an ethanolic solution of 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, PEG lipids, and ionizable cationic lipids was rapidly mixed with an aqueous solution (pH 4) containing mRNA and sgRNA (1:1 weight ratio) using an (in-line) mixer at a flow ratio (ethanol:aqueous phase) of 1:3. The N:P ratio between ionizable lipid and nucleic acid was maintained at 4-6 throughout the study.

The resulting LNP formulation was dialyzed against 1×PBS overnight, 0.2 μm sterile filtered and stored at 4°C until use. Particle size was in the range of 70-90 nm (Z-Ave, hydrodynamic diameter) and polydispersity index <0.2 as determined by dynamic light scattering (Malvern NanoZS Zetasizer). RNA encapsulation efficiency in LNP was measured by Quant-iT Ribogreen Assay (Life Technologies).

Cryo-TEM sample preparation and imaging

At 95% relative humidity, LNP samples (3-5 μl) were dispensed onto plasma-cleaned meshes (Quantifoil, R1.2/1.3 300 or 400 copper meshes) in a FEI Vitrobot chamber and left to stand for 30-60 s. The meshes were then blotted for 3 s with filter paper and immersed in liquid ethane cooled by liquid nitrogen. Cryo-EM imaging was performed on a FEI Talos F200C, operating at 200 kV accelerating voltage.

LNP contains a fusion molecule mRNA and sgRNA targeting the Pcsk9 gene in a weight ratio of 1:1 (Figure 3A). Lipid nanoparticles (LNP) are prepared using a mixture of ionizable lipids (20%-70%, molar ratio), PEGylated lipids (0%-30%, molar ratio), supporting lipids (30%-50%, molar ratio) and cholesterol (10%-50%, molar ratio) using a carefully designed impinging stream reactor or microfluidic device. By changing the ratio of ionizable lipids, the release kinetics of sgRNA and mRNA can be changed. When a higher ratio of ionizable lipids (molar ratio above 55%) is used, sgRNA is released much faster than mRNA. Transmission electron microscopy (TEM) images show that LNPs are spherical and nanosized particles (Figure 3B). Using dynamic light scattering (NanoSZ, Malvern), LNPs have uniform size (78.2±5.2nm, PDI<0.10) (Figure 3C).

Example 4: PCSK9 gene silencing in mice using LNPs to deliver mRNA and sgRNA encoding fusion molecules

Next, the use of the EPICAS system (also known as the "CRISPRoff" system) to silence Pcsk9 expression in vivo was tested. LNP was administered to C57CB/6J mice by tail vein injection (Figure 3E). Five days after injection, the mice were euthanized, and liver samples were obtained and processed to purify mRNA. RT-QPCR experiments were performed to evaluate the knockdown efficiency of the Pcsk9 gene in mice. The expression level of Pcsk9 in LNP-injected mice was significantly lower than that in the control group (Figure 3F), indicating the efficacy of the EPICAS system in silencing Pcsk9 gene expression in vivo.

To test whether LNPs could successfully deliver mRNA to mouse hepatocytes in vivo, LNPs containing luciferase mRNA were produced and injected into wild-type mice via intramuscular injection.

In vivo Luc mRNA delivery

To detect the in vivo distribution of Luc mRNA-LNP, 5 μg Luc mRNA was injected into female Balb/c mice (n=5) aged 6-8 weeks. At the specified detection time point, 0.2 ml D-luciferin (15 mg/ml in DPBS) was injected into the mice and imaged using the IVIS Lumina system (Perkin Elmer).

For in vitro imaging, 6-8 week old female BALB/c mice (n=2) were injected with 5 μg Luc mRNA. 12 hours later, animals were injected intraperitoneally (i.p.) with 0.2 mL D-luciferin (15 mg/ml in DPBS) and then reacted for 5 minutes. Tissues including heart, liver, spleen, lung and kidney were immediately collected and the fluorescence signal of each tissue was monitored by the IVIS Lumina system.

By in vivo fluorescence imaging, it is shown that LNP efficiently delivers luciferase mRNA to mouse liver cells (Fig. 3D). The results show that LNP can also efficiently deliver relatively large-sized mRNA to mouse liver by tail vein injection, as shown in the figure, almost all hepatocytes show tdTomato fluorescence (Fig. 6A). In addition, the luciferase mRNA delivered by LNP accumulates in mouse liver 6hr after injection (Fig. 6B), gradually decreases in mouse liver 12hr and 24hr after injection, and does not exist in liver 48hr after injection (Fig. 6C).

LNP treatment of mice

Mouse studies were approved and used for experiments by Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. and SLAC Laboratories. Ai9 (C57BL/6J genetic background) and C57BL/6J wild-type mice were housed in specific pathogen-free housing conditions and a 12h light/12h dark cycle. The use and care of animals complied with the guidelines of the Life Science Ethics Committee of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Male C57BL/6J mice were used for experiments at 8-10 weeks of age, mice were randomly assigned to various experimental groups, and data collection and analysis were performed in a blinded manner. LNPs were administered to mice in 200 μl PBS by tail vein injection. Mice were killed at designated time points, and liver samples and serum were obtained at autopsy for RNA extraction or serum biochemical analysis.

For CRISPRoff delivery in C57CB/6J adult wild-type mice, CRISPRoff mRNA and sgRNA (sgRNA 7 (SEQ ID NO: 33) and sgRNA 9 (SEQ ID NO: 35)) were delivered at a weight ratio of 1:1 by intravenous injection of LNPs (Figures 9A, 9B). Liver tissue was collected 7 days after injection for qPCR analysis. The expression of Pcsk9 was significantly reduced compared with control mice injected with PBS (Figure 6D). Further studies showed that doses of 1.5, 3.0, 6.0 and 10 mg/kg of LNPs containing CRISPRoff resulted in 76%, 93%, 97% and 98% inhibition of Pcsk9 expression, with a near-plateau effect at 3.0 mg/kg (Figure 6D). In CRISPRoff-treated mice, Pcsk9 protein levels in the blood were also significantly reduced, and its dose dependence was similar to that of Pcsk9 expression in liver tissue (Figure 6E). High levels of DNA methylation at the Pcsk9 gene promoter were observed at 3.0 and 6.0 mg/kg, while no abnormalities were found in blood chemistry (AST, ALT, ALP, and ALB) ( Figures 9C , 9D ).

Stable PCSK9 methylation and decreased expression after liver resection and regeneration

Mouse model of liver regeneration induced by partial hepatectomy (PHx)

Partial hepatectomy (PHx) in mice was performed as described previously (2). In brief, we used general anesthesia, a small upper midline incision, silk sutures to tie off the liver lobe to be removed, a warming pad and lamp, and subcutaneous saline injection to ensure minimal morbidity.

High-fat diet-induced hypercholesterolemia mouse model

High-fat diet mice were obtained from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. (Nanjing, China). The mice were housed under specific pathogen-free housing conditions and a 12 h light/12 h dark cycle and fed with a high-fat diet, a 60 kcal% saturated (lard) fat diet (HFD) obtained from Research Diets, Inc. (New Brunswick, NJ) for 24 weeks. Mice with blood LDL-c levels greater than 25 mg/dL were selected for the experiment.

Next, the persistence of CRISPRoff-induced Pcsk9 reduction at a dose of 6 mg/kg was evaluated. In the blood of CRISPRoff-treated mice, the protein expression levels of Pcsk9 decreased by 88%, 81%, 82% and 77% at 2, 4, 6 and 8 weeks after LNP injection, respectively, indicating that CRISPRofff persisted in silencing the target gene in vivo (Figure 6F). To further demonstrate the heritability of CRISPRoff-mediated epigenome editing, a liver resection experiment was performed to measure the gene silencing effect after liver regeneration. Specifically, mice were administered LNPs containing CRISPRoff on day 0 and underwent partial hepatectomy (PHx) (14) or sham surgery on day 7 (Figure 6G). Liver tissue samples after the PHx experiment were collected on day 14, when liver regeneration was almost complete (Figure 6G). The expression of Pcsk9 mRNA in the liver after PHx showed a similar reduction level as that in the sham group (93 +/- 11% and 92% +/- 9%, P < 0.0001, t-test) (Figure 6H). In addition, after PHx, CpG methylation at the Pcsk9 promoter was maintained at a high level (Figure 6I). In the late stage of PHx, since a large number of hepatocytes regenerate through cell division, these results indicate that CRISPRoff-mediated epigenome editing is heritable during cell division in vivo, which is beneficial for therapeutic design.

Sustained reduction of blood LDL in mice fed a high-fat diet

Serum LDL-C levels have been shown to increase with a high-fat diet (HFD) (15). The effects of epigenome editing on reducing Pcsk9 levels in mice fed an HFD were evaluated. Specifically, 6-week-old male C57BI/6J mice were fed an HFD for 6 months and then treated with LNPs or PBS encapsulated with CRISPRoff mRNA and Pcsk9-targeting sgRNA via tail vein injection (Figure 7A). At 7 and 14 days after injection, Pcsk9 levels in the blood were significantly reduced at doses of 4 mg/kg and 6 mg/kg compared to the PBS group (Figures 7B, 7C). In addition, at 4 mg/kg and 6 mg/kg, serum LDL-C levels decreased by approximately 44% and approximately 58%, respectively, 14 days after injection, and by approximately 43% and approximately 51%, respectively, 21 days after injection (Figure 7D). These results suggest that reduction of Pcsk9 by epigenome editing can efficiently and durably reduce serum LDL-C levels in HFD mice, which is advantageous in therapeutic design.

Precise silencing of PCSK9 by the EPICAS system without off-target effects

To investigate the potential off-target effects of epigenome editing, the specificity of CRISPRoff editing at both the transcriptome and genome levels was assessed. Seven days after LNP delivery of CRISPRoff mRNA and Pcsk9 targeting sgRNA, RNA-seq was performed on mouse liver tissue. In CRISPRoff treated mice, the expression levels of whole transcriptome genes were not significantly different from those of PBS treated mice, except that the expression level of the target gene Pcsk9 was silenced (Figures 8A and 10A). The expression levels of neighboring genes within the 1-Mb window from Pcsk9 did not show significant differences between the two groups of mice (Figure 8B). Several non-target transcripts with more than 2-fold changes (FDR<0.05) were observed in the CRISPRoff treated group (Figure 10B), but the DNA methylation levels on these non-target transcripts did not show significant differences between CRISP-off and PBS treated mice (Figure 10B). In addition, high-throughput whole genome bisulfite sequencing (WGBS) was performed to examine the off-target methylation of CpG on liver tissue. Except for Pcsk9, there was no significant difference in methylation levels at all CpG sites between the two groups of mice, indicating a dominant increase in DNA methylation at the Pcsk9 promoter in CRISPRoff-treated mice (Figure 8C). Detailed examination revealed that DNA methylation was upregulated only in the promoter region targeted by sgRNA, without spreading to the Pcsk9 gene body or neighboring genes (Figures 8D and 10C). Examination of genes at or near non-target differentially methylated regions did not reveal transcriptional differences (Figure 10D), indicating that nonspecific methylation differences had little effect on gene expression. We also compared methylation and gene expression levels at potential sgRNA-dependent off-target sites (with high sequence similarity to the target locus) and found no significant differences between CRISPRoff and PBS-treated mice (Figure 8E). Together, these results demonstrate that epigenetically mediated gene silencing induces few sgRNA-independent and sgRNA-dependent off-target effects in vivo.

In summary, the results demonstrated that the EPICAS (CRISPRoff) system can efficiently inhibit the expression of the target gene Pcsk9 in mouse liver by up to 98%, with an efficacy higher than that of existing drugs (statins, antibodies, and siRNA) and current gene editing technologies using CRISPR/Cas9 or base editors (8-12). Cutting-free epigenome editing using the EPICAS (CRISPRoff) system reduces the potential risk of unnecessary DNA repair-mediated editing at the target locus, which is beneficial for human therapeutic design. It also does not induce detectable off-target DNA methylation and changes in gene expression. This CRISPRoff-induced methylation can be reversed by CRISPR-mediated demethylation tools (4). Notably, using transient delivery of CRISProff mRNA, CRISPRoff-dependent downregulation of the target gene persists after multiple rounds of cell division. In vivo delivery of gene editing tools using LNPs may be better than AAV because long-term expression of editing tools caused by AAV delivery is neither necessary nor desirable. Transient LNP-mediated mRNA delivery avoids off-target effects caused by the prolonged presence of the editor and other side effects of AAV, such as immune responses and genomic integration of the editing tool (16). Finally, epigenome editing-induced Pcsk9 silencing and blood LDL-C reduction were robust and durable, providing a potential therapeutic strategy for the treatment of FH. Such approaches may also be applied to the treatment of other chronic diseases.

Example 5: Monkey PCSK9 gene silencing in monkey cells

Various sgRNAs were designed to target the regions within 250 bp upstream and downstream of the transcription start site (TSS) of the monkey Pcsk9 gene ( FIG. 4A ).

Each sgRNA plasmid was co-transfected with a catalytic protein (DNMT3A-DNMT3L-dCas9-KRAB) plasmid into monkey cells. RT-QPCR experiments were performed to evaluate the mRNA expression level of monkey Pcsk9. Three of the five sgRNAs tested showed significant downregulation of Pcsk9 expression in monkey cells (Figure 4B). Cells transfected with S2, S8 or S9 sgRNAs resulted in approximately 90% downregulation of monkey Pcks9.

Example 6: Human PCSK9 gene silencing in human cell lines

A reporter cell line was constructed to test the efficiency of PCKS9 gene silencing in human cell lines. A plasmid with a CMV promoter-driven cassette was constructed, wherein the cassette had the following elements in the 5' to 3' direction: 5'-pCMV--300bp-TSS-+300bp-PCSK9 exon 1-2A-GFP-3'. In this reporter system, the CMV promoter drives the expression of PCSK9 and GFP fluorescence. If PCSK9 is silenced, the transcription of GFP is terminated. The reporter plasmid was transfected into HEK293T cells together with the PiggyBac transposase (PBase) plasmid. According to the expression of GFP fluorescence, cells with successfully integrated reporter gene cassettes were sorted by FACS (Fig. 5A).

109 sgRNAs were designed to target the regions 300bp upstream and 300bp downstream of the PCSK9 TSS. Plasmids were constructed to encode each sgRNA. Each sgRNA plasmid was co-transfected with a plasmid encoding a fusion molecule into a human reporter cell line. The reduction in the average GFP intensity rate at 72h and 120h after transfection was analyzed. The overall reduction in the GFP intensity rate indicates the sensitivity of the reporter system (Figure 5B). The reduced GFP intensity rate after transfection was maintained for 120h. Compared with 72h after transfection, many sgRNAs showed much lower GFP intensity rates at 120h. These results show the efficacy and persistence of the EPICAS system in human cell lines.

Next, the experiment was repeated using each sgRNA, and the mean GFP intensity ratio was measured 72 h after transfection for comparison (Figure 5C). More than half of the designed sgRNAs showed a significant decrease in the mean fluorescence intensity ratio, indicating that the EPICAS system can induce targeted knockdown of PCSK9 expression in human cells.

Next, the use of the EPICAS system in silencing endogenous PCSK9 expression was tested in the human Hep3B cell line. The plasmid encoding the fusion molecule was co-transfected with various sgRNA plasmids into Hep3B cells. 48h after transfection, the mRNA expression level of human PCSK9 was measured by RT-PCR. The six sgRNAs resulted in the highest decrease in PCSK9 expression levels (approximately >65%). These sgRNAs also resulted in a decrease in the fluorescence intensity rate in the reporter human cell line by approximately >50% (Figure 5D). These results show that the EPICAS system can silence the expression of endogenous PCSK9 in human cells efficiently and for a long time.

In summary, these results show that the EPICAS system successfully silenced PCSK9 expression in both mouse and human cells, and efficiently and persistently supported silencing PCSK9 gene expression through epigenetic editing. LNP has been successfully used to deliver the EPICAS system in vivo. Therefore, the LNP formulation of the EPICAS system can be used to treat PCSK9-related diseases such as atherosclerotic cardiovascular disease by reducing low-density lipoprotein cholesterol (LDL) by reducing PCSK9 expression.

Other embodiments of the present disclosure include the following:

Embodiment 1. A method for reducing or eliminating the expression of a proprotein convertase subtilisin/Kexin type 9 (PCSK9) gene product in a cell, the method comprising the step of introducing the following substances into the cell:

A fusion molecule comprising at least one DNA binding protein and at least one gene expression regulator, or a nucleic acid sequence encoding the fusion molecule,

wherein the gene expression regulator provides modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element,

Thereby reducing or eliminating the expression of the PCSK9 gene product in the cell.

Embodiment 2. An in vivo method for reducing or eliminating the expression of a PCSK9 gene product in a subject, the method comprising the step of introducing into cells of the subject:

A fusion molecule comprising at least one DNA binding protein and at least one gene expression regulator, or a nucleic acid sequence encoding the fusion molecule,

wherein the gene expression regulator provides modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element,

Thereby reducing or eliminating the expression of the PCSK9 gene product in the subject.

Embodiment 3. A method for reducing low-density lipoprotein (LDL) cholesterol in a subject, the method comprising the step of introducing into cells of the subject:

A fusion molecule comprising at least one DNA binding protein and at least one gene expression regulator, or a nucleic acid sequence encoding the fusion molecule,

wherein the gene expression regulator provides modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element,

Thereby reducing LDL cholesterol in said subject.

Embodiment 4. A method for treating or alleviating the symptoms of a PCSK9-related disease in a subject, the method comprising the step of introducing the following substances into the cells of the subject:

A fusion molecule comprising at least one DNA binding protein and at least one gene expression regulator, or a nucleic acid sequence encoding the fusion molecule,

wherein the gene expression regulator provides modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element,

Thereby treating or alleviating the symptoms of the PCSK9-related disease in the subject.

Embodiment 5. A method for expanding a cell population with reduced expression of a PCSK9 gene product, the method comprising the following steps:

i) introducing a fusion molecule comprising at least one DNA binding protein and at least one gene expression regulator or a nucleic acid sequence encoding the fusion molecule into a plurality of cells,

wherein the gene expression regulator provides modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element;

ii) expanding the plurality of cells to produce a plurality of modified cells having reduced expression of a PCSK9 gene product,

wherein the expression of the PCSK9 gene product of the plurality of modified cells is reduced by at least 50%, at least 60%, at least 70%, at least 80% or at least 90% relative to cells into which the fusion molecule or the nucleic acid sequence has not been introduced, and

Wherein the cell is a hepatocyte.

Embodiment 6. The method according to embodiment 5, wherein the reduced expression of the PCSK9 gene product is a transient reduction.

Embodiment 7. The method according to embodiment 5, wherein the reduced expression of the PCSK9 gene product is a stable reduction.

Embodiment 8. A method according to any one of embodiments 1-7, wherein the PCSK9 regulatory element is a core promoter, a proximal promoter, a distal enhancer, a silencer, an insulator element, a boundary element or a locus control region.

Embodiment 9. A method according to any one of embodiments 1-8, wherein the modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element is located within about 100bp, about 200bp, about 300bp, about 400bp, about 500bp, about 600bp, about 700bp, about 800bp, about 900bp, about 1000bp, about 1100bp, about 1200bp, about 1300bp, about 1400bp or about 1500bp upstream of the transcription start site of the PCSK9 gene.

Embodiment 10. A method according to embodiment 9, wherein the modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element is located within 1000 bp upstream of the transcription start site of the PCSK9 gene.

Embodiment 11. A method according to embodiment 9, wherein the modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element is located within 300 bp upstream of the transcription start site of the PCSK9 gene.

Embodiment 12. A method according to any one of embodiments 1-11, wherein the modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element is located within about 100bp, about 200bp, about 300bp, about 400bp, about 500bp, about 600bp, about 700bp, about 800bp, about 900bp, about 1000bp, about 1100bp, about 1200bp, about 1300bp, about 1400bp or about 1500bp downstream of the transcription start site of the PCSK9 gene.

Embodiment 13. A method according to embodiment 12, wherein the modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element is located within about 300 bp downstream of the transcription start site of the PCSK9 gene.

Embodiment 14. A method according to any one of embodiments 1-8, wherein the modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element is located within 1000 bp upstream of the transcription start site of the PCSK9 gene and within 300 bp downstream of the transcription start site.

Embodiment 15. A method according to any one of embodiments 1-14, wherein the modification of at least one nucleotide is DNA methylation.

Embodiment 16. A method according to any one of embodiments 1-15, wherein the at least one gene expression regulator comprises a DNA methyltransferase (DNMT), a DNA demethylase, a histone methyltransferase, a histone demethylase or a portion thereof.

Embodiment 17. The method of embodiment 16, wherein the at least one gene expression regulator comprises a DNA methyltransferase (DNMT) or a portion thereof.

Embodiment 18. A method according to embodiment 17, wherein the DNA methyltransferase is DNMT3A, DNMT3B, DNMT3L, DNMT1 or DNMT2.

Embodiment 19. A method according to embodiment 18, wherein the DNMT3A comprises the amino acid sequence of SEQ ID NO: 23.

Embodiment 20. The method according to embodiment 18, wherein the DNMT3L comprises the amino acid sequence of SEQ ID NO: 24.

Embodiment 21. A method according to any one of embodiments 1-20, wherein the at least one gene expression regulator comprises a zinc finger protein-based transcription factor or a portion thereof.

Embodiment 22. A method according to embodiment 21, wherein the zinc finger protein-based transcription factor is a Kruppel-associated repression box (KRAB).

Embodiment 23. A method according to embodiment 22, wherein the KRAB comprises the amino acid sequence of SEQ ID NO:22.

Embodiment 24. A method according to any one of embodiments 1-23, wherein the at least one gene expression regulator comprises a DNA methyltransferase or a portion thereof and a zinc finger protein-based transcription factor or a portion thereof.

Embodiment 25. A method according to embodiment 24, wherein the DNA methyltransferase is selected from DNMT3A and DNMT3L and a combination thereof, and the zinc finger protein-based transcription factor is KRAB.

Embodiment 26. A method according to any one of embodiments 1-25, wherein at least one of the DNA binding proteins is Cas9, dCas9, Cpf1, zinc finger nuclease (ZNF), transcription activator-like effector nuclease (TALEN), homing endonuclease, dCas9-FokI nuclease or MegaTal nuclease.

Embodiment 27. A method according to embodiment 26, wherein at least one DNA binding protein is dCas9.

Embodiment 28. The method of embodiment 27, wherein the dCas9 comprises Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, Campylobacter jejuni dCas9, Corynebacterium diphtheria dCas9, Eubacterium ventriosum dCas9, Streptococcus pasteurianus dCas9, Lactobacillus farciminis dCas9, Sphaerochaeta globus dCas9, Azospirillum (e.g., strain B510) dCas9, Gluconacetobacter diazotrophicus dCas9, Neisseria cinerea dCas9, Roseburia enterica dCas9, or any combination thereof. intestinalis) dCas9, Parvibaculum lavamentivorans dCas9, Nitratifractor salsuginis (e.g., strain DSM16511) dCas9, Campylobacter lari (e.g., strain CF89-12) dCas9, Streptococcus thermophilus (e.g., strain LMD-9) dCas9.

Embodiment 29. A method according to embodiment 27, wherein the dCas9 comprises the amino acid sequence of SEQ ID NO: 1.

Embodiment 30. A method according to any one of embodiments 1-29, wherein the fusion molecule comprises at least one gene expression regulator fused to the C-terminus, N-terminus, or both of the at least one DNA binding protein.

Embodiment 31. A method according to embodiment 30, wherein the at least one gene expression regulator is directly fused to the at least one DNA binding protein.

Embodiment 32. A method according to embodiment 30, wherein the at least one gene expression regulator is indirectly fused to the at least one DNA binding protein via a non-regulatory agent, a second regulator, or a linker.

Embodiment 33. A method according to any one of embodiments 30-32, wherein the fusion molecule comprises dCas9 fused to KRAB at the C-terminal end and to DNMT3A and DNMT3L at the N-terminal end.

Embodiment 34. A method according to embodiment 33, wherein the fusion molecule comprises the amino acid sequence of SEQ ID NO:97.

Embodiment 35. A method according to any one of embodiments 1-34, wherein the fusion molecule further comprises at least one nuclear localization sequence.

Embodiment 36. A method according to embodiment 35, wherein the at least one nuclear localization sequence is directly fused to the C-terminus, N-terminus, or both of the at least one DNA binding protein.

Embodiment 37. A method according to embodiment 35, wherein the at least one nuclear localization sequence is indirectly fused to the C-terminus, N-terminus, or both of the at least one DNA binding protein via a linker.

Embodiment 38. A method according to any one of embodiments 1-37, wherein the nucleic acid sequence encoding the fusion molecule is deoxyribonucleic acid (DNA).

Embodiment 39. A method according to any one of embodiments 1-37, wherein the nucleic acid sequence encoding the fusion molecule is messenger ribonucleic acid (mRNA).

Embodiment 40. A method according to any one of embodiments 1-39, further comprising the step of introducing at least one single guide RNA (sgRNA) or a DNA encoding the sgRNA, wherein the sgRNA is complementary to a DNA sequence near the PCSK9 gene and/or within the PCSK9 regulatory element, thereby targeting the fusion molecule to the PCSK9 gene or the PCSK9 regulatory element.

Embodiment 41. A method according to embodiment 40, wherein the sgRNA comprises a nucleic acid sequence of SEQ ID NO: 27-95 or 98-108.

Embodiment 42. A method according to any one of embodiments 1-41, wherein the fusion molecule is formulated in a liposome or lipid nanoparticle.

Embodiment 43. A method according to any one of embodiments 40-41, wherein the fusion molecule and the sgRNA are formulated in liposomes or lipid nanoparticles.

Embodiment 44. A method according to embodiment 43, wherein the fusion molecule and the sgRNA are formulated in the same liposome or lipid nanoparticle.

Embodiment 45. A method according to embodiment 43, wherein the fusion molecule and the sgRNA are formulated in different liposomes or lipid nanoparticles.

Embodiment 46. A method according to any one of embodiments 42-45, wherein the liposomes or lipid nanoparticles contain ionizable lipids (20%-70%, molar ratio), PEGylated lipids (0%-30%, molar ratio), supporting lipids (5%-50%, molar ratio) and cholesterol (10%-50%, molar ratio).

Embodiment 47. A method according to embodiment 46, wherein the ionizable lipid is selected from pH-responsive ionizable lipids, heat-responsive ionizable lipids and light-responsive ionizable lipids.

Embodiment 48. A method according to any one of embodiments 1-41, wherein the fusion molecule is formulated in an AAV vector.

Embodiment 49. A method according to any one of embodiments 40-41, wherein the fusion molecule and the sgRNA are formulated in an AAV vector.

Embodiment 50. A method according to embodiment 49, wherein the fusion molecule and the sgRNA are formulated in the same AAV vector.

Embodiment 51. A method according to embodiment 49, wherein the fusion molecule and the sgRNA are formulated in different AAV vectors.

Embodiment 52. A method according to any one of embodiments 1-51, wherein the fusion molecule is delivered to the cell by local injection, systemic infusion, or a combination thereof.

Embodiment 53. A method according to any one of embodiments 2-4 and 8-52, wherein the subject is human.

Embodiment 54. A method according to any one of embodiments 4 and 8-53, wherein the PCSK9-related disease is atherosclerotic cardiovascular disease.

Embodiment 55. A method according to any one of embodiments 4 and 8-53, wherein the PCSK9-related disease is hypercholesterolemia.

Embodiment 56. A method according to any one of embodiments 1-55, wherein the cell is a hepatocyte.

Embodiment 57. An sgRNA comprising the nucleic acid sequence of any one of SEQ ID NOs: 27-95 or 98-108.

Embodiment 58. A DNA sequence encoding the sgRNA according to embodiment 54.

Embodiment 59. A pharmaceutical composition comprising: a fusion molecule comprising at least one DNA binding protein and at least one gene expression regulator, or a nucleic acid sequence encoding the fusion molecule,

wherein the fusion molecule targets a genomic region near the PCSK9 gene and/or within a PCSK9 regulatory element,

wherein the at least one gene expression regulator provides for modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element,

wherein the at least one gene expression regulator comprises a DNA methyltransferase (DNMT), a DNA demethylase, a histone methyltransferase, a histone demethylase or a portion thereof, or a zinc finger protein-based transcription factor or a portion thereof, or a combination thereof, and

wherein the at least one DNA binding protein is Cas9, dCas9, Cpf1, zinc finger nuclease (ZNF), transcription activator-like effector nuclease (TALEN), homing endonuclease, dCas9-FokI nuclease or MegaTal nuclease.

Embodiment 60. A pharmaceutical composition according to embodiment 59, wherein the PCSK9 regulatory element is a transcription start site, a core promoter, a proximal promoter, a distal enhancer, a silencer, an insulator element, a boundary element or a locus control region.

Embodiment 61. A pharmaceutical composition according to any one of embodiments 59-60, wherein the modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element is located within about 100bp, about 200bp, about 300bp, about 400bp, about 500bp, about 600bp, about 700bp, about 800bp, about 900bp, about 1000bp, about 1100bp, about 1200bp, about 1300bp, about 1400bp or about 1500bp upstream of the transcription start site of the PCSK9 gene.

Embodiment 62. A pharmaceutical composition according to embodiment 61, wherein the modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element is located within 1000 bp upstream of the transcription start site of the PCSK9 gene.

Embodiment 63. A pharmaceutical composition according to embodiment 61, wherein the modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element is located within 300 bp upstream of the transcription start site of the PCSK9 gene.

Embodiment 64. A pharmaceutical composition according to any one of embodiments 59-63, wherein the modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element is located within about 100bp, about 200bp, about 300bp, about 400bp, about 500bp, about 600bp, about 700bp, about 800bp, about 900bp, about 1000bp, about 1100bp, about 1200bp, about 1300bp, about 1400bp or about 1500bp downstream of the transcription start site of the PCSK9 gene.

Embodiment 65. A pharmaceutical composition according to embodiment 64, wherein the modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element is located within about 300 bp downstream of the transcription start site of the PCSK9 gene.

Embodiment 66. A pharmaceutical composition according to any one of embodiments 59-65, wherein the modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element is located within 1000 bp upstream of the transcription start site of the PCSK9 gene and within 300 bp downstream of the transcription start site.

Embodiment 67. A pharmaceutical composition according to any one of embodiments 59-66, wherein the modification of at least one nucleotide is DNA methylation.

Embodiment 68. A pharmaceutical composition according to embodiments 59-67, wherein the at least one gene expression regulator comprises a DNA methyltransferase (DNMT) or a portion thereof.

Embodiment 69. A pharmaceutical composition according to embodiment 68, wherein the DNA methyltransferase is DNMT3A, DNMT3B, DNMT3L, DNMT1 or DNMT2.

Embodiment 70. A pharmaceutical composition according to embodiment 69, wherein the DNMT3A comprises the amino acid sequence of SEQ ID NO: 23.

Embodiment 71. A pharmaceutical composition according to embodiment 69, wherein the DNMT3L comprises the amino acid sequence of SEQ ID NO: 24.

Embodiment 72. A pharmaceutical composition according to any one of embodiments 59-71, wherein the at least one gene expression regulator comprises a zinc finger protein-based transcription factor or a portion thereof.

Embodiment 73. A pharmaceutical composition according to embodiment 72, wherein the zinc finger protein-based transcription factor is Kruppel-associated repression box (KRAB).

Embodiment 74. The pharmaceutical composition of embodiment 73, wherein the KRAB comprises the amino acid sequence of SEQ ID NO: 22.

Embodiment 75. A pharmaceutical composition according to any one of embodiments 59-74, wherein the at least one gene expression regulator comprises a DNA methyltransferase or a portion thereof and a zinc finger protein-based transcription factor or a portion thereof.

Embodiment 76. A pharmaceutical composition according to embodiment 75, wherein the DNA methyltransferase is selected from DNMT3A and DNMT3L and a combination thereof, and the zinc finger protein-based transcription factor is KRAB.

Embodiment 77. A pharmaceutical composition according to any one of embodiments 59-76, wherein the at least one DNA binding protein is Cas9, dCas9, Cpf1, zinc finger nuclease (ZNF), transcription activator-like effector nuclease (TALEN), homing endonuclease, dCas9-FokI nuclease or MegaTal nuclease.

Embodiment 78. A pharmaceutical composition according to embodiment 77, wherein the at least one DNA binding protein is dCas9.

Embodiment 79. A pharmaceutical composition according to embodiment 78, wherein the dCas9 comprises Staphylococcus aureus dCas9, Streptococcus pyogenes dCas9, Campylobacter jejuni dCas9, Corynebacterium diphtheria dCas9, Eubacterium ventriosum dCas9, Streptococcus pasteurianus dCas9, Lactobacillus farciminis dCas9, Sphaerochaeta globus dCas9, Azospirillum (e.g., strain B510) dCas9, Gluconacetobacter diazotrophicus dCas9, Neisseria cinerea dCas9, Roseburia enterica dCas9, or any combination thereof. intestinalis) dCas9, Parvibaculum lavamentivorans dCas9, Nitratifractor salsuginis (e.g., strain DSM16511) dCas9, Campylobacter lari (e.g., strain CF89-12) dCas9, Streptococcus thermophilus (e.g., strain LMD-9) dCas9.

Embodiment 80. A pharmaceutical composition according to embodiment 78, wherein the dCas9 comprises the amino acid sequence of SEQ ID NO: 1.

Embodiment 81. A pharmaceutical composition according to any one of embodiments 59-80, wherein the fusion molecule comprises the at least one gene expression regulator fused to the C-terminus, N-terminus, or both of the at least one DNA binding protein.

Embodiment 82. A pharmaceutical composition according to embodiment 81, wherein the at least one gene expression regulator is directly fused to the at least one DNA binding protein.

Embodiment 83. A pharmaceutical composition according to embodiment 81, wherein the at least one gene expression regulator is indirectly fused to the at least one DNA binding protein through a non-regulatory agent, a second regulator or a linker.

Embodiment 84. A pharmaceutical composition according to any one of embodiments 81-83, wherein the fusion molecule comprises dCas9 fused to KRAB at the C-terminal end and to DNMT3A and DNMT3L at the N-terminal end.

Embodiment 85. A pharmaceutical composition according to embodiment 84, wherein the fusion molecule comprises the amino acid sequence of SEQ IDNO:97.

Embodiment 86. A pharmaceutical composition according to any one of embodiments 59-85, wherein the fusion molecule further comprises at least one nuclear localization sequence.

Embodiment 87. A pharmaceutical composition according to embodiment 86, wherein the at least one nuclear localization sequence is directly fused to the C-terminus, N-terminus, or both, of the at least one DNA binding protein.

Embodiment 88. A pharmaceutical composition according to embodiment 86, wherein the at least one nuclear localization sequence is indirectly fused to the C-terminus, N-terminus, or both of the at least one DNA binding protein via a linker.

Embodiment 89. A pharmaceutical composition according to any one of embodiments 59-88, wherein the nucleic acid sequence encoding the fusion molecule is deoxyribonucleic acid (DNA).

Embodiment 90. A pharmaceutical composition according to any one of embodiments 59-88, wherein the nucleic acid sequence encoding the fusion molecule is messenger ribonucleic acid (mRNA).

Embodiment 91. A pharmaceutical composition according to any one of embodiments 59-90, further comprising at least one single guide RNA (sgRNA) complementary to a DNA sequence near the PCSK9 gene and/or within the PCSK9 regulatory element.

Embodiment 92. A pharmaceutical composition according to embodiment 91, wherein the sgRNA comprises a nucleic acid sequence of SEQ ID NO: 27-95 or 98-108.

Embodiment 93. A pharmaceutical composition according to any one of embodiments 59-92, wherein the fusion molecule is packaged in a liposome or a lipid nanoparticle.

Embodiment 94. A pharmaceutical composition according to any one of embodiments 91-92, wherein the fusion molecule and the sgRNA are packaged in liposomes or lipid nanoparticles.

Embodiment 95. A pharmaceutical composition according to embodiment 94, wherein the fusion molecule and the sgRNA are packaged in the same liposome or lipid nanoparticle.

Embodiment 95. A pharmaceutical composition according to embodiment 94, wherein the fusion molecule and the sgRNA are packaged in different liposomes or lipid nanoparticles.

Embodiment 97. A pharmaceutical composition according to any one of embodiments 93-96, wherein the liposomes or lipid nanoparticles contain ionizable lipids (20%-70%, molar ratio), PEGylated lipids (0%-30%, molar ratio), supporting lipids (5%-50%, molar ratio) and cholesterol (10%-50%, molar ratio).

Embodiment 98. A pharmaceutical composition according to any one of embodiments 93-97, wherein the ionizable lipid is selected from pH-responsive ionizable lipids, thermo-responsive ionizable lipids and light-responsive ionizable lipids.

Embodiment 99. A pharmaceutical composition according to any one of embodiments 59-92, wherein the fusion molecule is packaged in an AAV vector.

Embodiment 100. A pharmaceutical composition according to any one of embodiments 91-92, wherein the fusion molecule and the sgRNA are packaged in an AAV vector.

Embodiment 101. A pharmaceutical composition according to embodiment 100, wherein the fusion molecule and the sgRNA are packaged in the same AAV vector.

Embodiment 102. A pharmaceutical composition according to embodiment 100, wherein the fusion molecule and the sgRNA are packaged in different AAV vectors.

Claims (42)

1. A method for reducing or eliminating the expression of a proprotein convertase subtilisin/kexin type 9 (PCSK9) gene product in a cell, the method comprising the step of introducing into the cell: A fusion molecule comprising at least one DNA binding protein and at least one gene expression regulator, or a nucleic acid sequence encoding the fusion molecule, wherein the gene expression regulator provides modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element, Thereby reducing or eliminating the expression of the PCSK9 gene product in the cell. 2. An in vivo method for reducing or eliminating the expression of a PCSK9 gene product in a subject, the method comprising the step of introducing into the cells of the subject: A fusion molecule comprising at least one DNA binding protein and at least one gene expression regulator, or a nucleic acid sequence encoding the fusion molecule, wherein the gene expression regulator provides modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element, Thereby reducing or eliminating the expression of the PCSK9 gene product in the subject. 3. A method for reducing low-density lipoprotein (LDL) cholesterol in a subject, the method comprising the step of introducing into cells of the subject: A fusion molecule comprising at least one DNA binding protein and at least one gene expression regulator, or a nucleic acid sequence encoding the fusion molecule, wherein the gene expression regulator provides modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element, Thereby reducing LDL cholesterol in said subject. 4. A method for treating or alleviating the symptoms of a PCSK9-related disease in a subject, the method comprising the step of introducing into the subject's cells: A fusion molecule comprising at least one DNA binding protein and at least one gene expression regulator, or a nucleic acid sequence encoding the fusion molecule, wherein the gene expression regulator provides modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element, Thereby treating or alleviating the symptoms of the PCSK9-related disease in the subject. 5. A method for amplifying a cell population with reduced expression of a PCSK9 gene product, the method comprising the steps of: i) introducing a fusion molecule comprising at least one DNA binding protein and at least one gene expression regulator or a nucleic acid sequence encoding the fusion molecule into a plurality of cells, wherein the gene expression regulator provides modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element; ii) expanding the plurality of cells to produce a plurality of modified cells having reduced expression of a PCSK9 gene product, wherein the expression of the PCSK9 gene product of the plurality of modified cells is reduced by at least 50%, at least 60%, at least 70%, at least 80% or at least 90% relative to cells into which the fusion molecule or the nucleic acid sequence has not been introduced, and Wherein the cell is a hepatocyte. 6. The method of any one of claims 1-5, wherein the PCSK9 regulatory element is a core promoter, a proximal promoter, a distal enhancer, a silencer, an insulator element, a boundary element, or a locus control region. 7. The method according to any one of claims 1 to 6, wherein the modification of the at least one nucleotide is DNA methylation. 8. The method of any one of claims 1-7, wherein the at least one gene expression regulator comprises a DNA methyltransferase (DNMT), a DNA demethylase, a histone methyltransferase, a histone demethylase, or a portion thereof. 9. The method of claim 8, wherein the DNMT is DNMT3A, which comprises the amino acid sequence of SEQ ID NO: 23. 10. The method of claim 8, wherein the DNMT is DNMT3L comprising the amino acid sequence of SEQ ID NO: 24. 11. The method of any one of claims 1-10, wherein the at least one gene expression regulator comprises a zinc finger protein-based transcription factor or a portion thereof. 12. The method of claim 11, wherein the zinc finger protein-based transcription factor is a Kruppel-associated repression cassette (KRAB). 13. The method of claim 12, wherein the KRAB comprises the amino acid sequence of SEQ ID NO: 22. 14. The method of any one of claims 1-13, wherein the at least one gene expression regulator comprises a DNA methyltransferase or a portion thereof and a zinc finger protein-based transcription factor or a portion thereof. 15. The method of claim 14, wherein the DNA methyltransferase is selected from DNMT3A and DNMT3L and combinations thereof, and the zinc finger protein-based transcription factor is KRAB. 16. The method of any one of claims 1-15, wherein the at least one DNA binding protein is Cas9, dCas9, Cpf1, zinc finger nuclease (ZNF), transcription activator-like effector nuclease (TALEN), homing endonuclease, dCas9-FokI nuclease, or MegaTal nuclease. 17. The method of claim 16, wherein the at least one DNA binding protein is dCas9. 18. The method of claim 17, wherein the dCas9 comprises the amino acid sequence of SEQ ID NO: 1. 19. The method of any one of claims 1-18, wherein the fusion molecule comprises dCas9 fused to KRAB on the C-terminal end and to DNMT3A and DNMT3L on the N-terminal end. 20. The method of any one of claims 1-19, wherein the fusion molecule comprises the amino acid sequence of SEQ ID NO: 97. 21. The method according to any one of claims 1-20, further comprising the step of introducing at least one single guide RNA (sgRNA) or a DNA encoding the sgRNA, wherein the sgRNA is complementary to a DNA sequence near the PCSK9 gene and/or within the PCSK9 regulatory element, thereby targeting the fusion molecule to the PCSK9 gene or PCSK9 regulatory element. 22. The method of claim 21, wherein the sgRNA comprises a nucleic acid sequence of SEQ ID NO: 27-95 or 98-108. 23. An sgRNA comprising the nucleic acid sequence of any one of SEQ ID NOs: 27-95 or 98-108. 24. A DNA sequence encoding the sgRNA according to claim 23. 25. A pharmaceutical composition comprising: a fusion molecule comprising at least one DNA binding protein and at least one gene expression regulator, or a nucleic acid sequence encoding the fusion molecule, wherein the fusion molecule targets a genomic region near the PCSK9 gene and/or within a PCSK9 regulatory element, wherein the at least one gene expression regulator provides for modification of at least one nucleotide near the PCSK9 gene and/or within the PCSK9 regulatory element, wherein the at least one gene expression regulator comprises a DNA methyltransferase (DNMT), a DNA demethylase, a histone methyltransferase, a histone demethylase or a portion thereof, or a zinc finger protein-based transcription factor or a portion thereof, or a combination thereof, and wherein the at least one DNA binding protein is Cas9, dCas9, Cpf1, zinc finger nuclease (ZNF), transcription activator-like effector nuclease (TALEN), homing endonuclease, dCas9-FokI nuclease or MegaTal nuclease. 26. The pharmaceutical composition of claim 25, wherein the PCSK9 regulatory element is a transcription start site, a core promoter, a proximal promoter, a distal enhancer, a silencer, an insulator element, a boundary element, or a locus control region. 27. A pharmaceutical composition according to any one of claims 25-26, wherein the modification of at least one nucleotide is DNA methylation. 28. The pharmaceutical composition of claims 25-27, wherein the at least one gene expression regulator comprises a DNA methyltransferase (DNMT) or a portion thereof. 29. The pharmaceutical composition according to claim 28, wherein the DNMT is DNMT3A, which comprises the amino acid sequence of SEQ ID NO: 23. 30. The pharmaceutical composition of claim 28, wherein the DNMT is DNMT3L, which comprises the amino acid sequence of SEQ ID NO: 24. 31. The pharmaceutical composition of any one of claims 25-30, wherein the at least one gene expression regulator comprises a zinc finger protein-based transcription factor or a portion thereof. 32. The pharmaceutical composition of claim 31, wherein the zinc finger protein-based transcription factor is a Kruppel-associated repression cassette (KRAB). 33. The pharmaceutical composition of claim 32, wherein the KRAB comprises the amino acid sequence of SEQ ID NO: 22. 34. The pharmaceutical composition of any one of claims 25-33, wherein the at least one gene expression regulator comprises a DNA methyltransferase or a portion thereof and a zinc finger protein-based transcription factor or a portion thereof. 35. The pharmaceutical composition of claim 34, wherein the DNA methyltransferase is selected from DNMT3A and DNMT3L and combinations thereof, and the zinc finger protein-based transcription factor is KRAB. 36. A pharmaceutical composition according to any one of claims 25-35, wherein the at least one DNA binding protein is Cas9, dCas9, Cpf1, zinc finger nuclease (ZNF), transcription activator-like effector nuclease (TALEN), homing endonuclease, dCas9-FokI nuclease or MegaTal nuclease. 37. The pharmaceutical composition of claim 36, wherein the at least one DNA binding protein is dCas9. 38. The pharmaceutical composition of claim 37, wherein the dCas9 comprises the amino acid sequence of SEQ ID NO: 1. 39. The pharmaceutical composition of claims 25-38, wherein the fusion molecule comprises dCas9 fused to KRAB on the C-terminal end and to DNMT3A and DNMT3L on the N-terminal end. 40. The pharmaceutical composition of claim 39, wherein the fusion molecule comprises the amino acid sequence of SEQ ID NO: 97. 41. The pharmaceutical composition according to any one of claims 25-40, further comprising at least one single guide RNA (sgRNA) complementary to a DNA sequence near the PCSK9 gene and/or within a PCSK9 regulatory element. 42. The pharmaceutical composition according to claim 41, wherein the sgRNA comprises a nucleic acid sequence of SEQ ID NO: 27-95 or 98-108.